RESUMO
Prostate cancer (PCa), one of the most common malignancies in men, typically responds to initial treatment, but resistance to therapy often leads to metastases and death. The dehydrogenase/reductase 7 (DHRS7, SDR34C1) is an "orphan" enzyme without known physiological function. DHRS7 was previously found to be decreased in higher-stage PCa, and siRNA-mediated knockdown increased the aggressiveness of LNCaP cells. To further explore the role of DHRS7 in PCa, we analyzed the proteome of LNCaP cells following DHRS7 knockdown to assess potentially altered pathways. Although DHRS7 is able to inactivate 5α-dihydrotestosterone, DHRS7 knockdown did not affect androgen receptor (AR) target gene expression, and its effect on PCa cells seems to be androgen-independent. Importantly, proteome analyses revealed increased expression of epidermal growth factor receptor (EGFR), which was confirmed by RT-qPCR and Western blotting. Comparison of AR-positive LNCaP with AR-negative PC-3 and DU145 PCa cell lines revealed a negative correlation between DHRS7 and EGFR expression. Conversely, EGFR knockdown enhanced DHRS7 expression in these cells. Importantly, analysis of patient samples revealed a negative correlation between DHRS7 and EGFR expression, both at the mRNA and protein levels, and DHRS7 expression correlated positively with patient survival rates. These results suggest a protective role for DHRS7 in PCa.
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BACKGROUND: Gliomas are the most frequent and aggressive malignancies of the central nervous system. Decades of molecular analyses have demonstrated that gliomas accumulate genetic alterations that culminate in enhanced activity of receptor tyrosine kinases and downstream mediators. While the genetic alterations, like gene amplification or loss, have been well characterized, little information exists about changes in the proteome of gliomas of different grades. METHODS: We performed unbiased quantitative proteomics of human glioma biopsies by mass spectrometry followed by bioinformatic analysis. FINDINGS: Various pathways were found to be up- or downregulated. In particular, endocytosis as pathway was affected by a vast and concomitant reduction of multiple machinery components involved in initiation, formation, and scission of endocytic carriers. Both clathrin-dependent and -independent endocytosis were changed, since not only clathrin, AP-2 adaptins, and endophilins were downregulated, but also dynamin that is shared by both pathways. The reduction of endocytic machinery components caused increased receptor cell surface levels, a prominent phenotype of defective endocytosis. Analysis of additional biopsies revealed that depletion of endocytic machinery components was a common trait of various glioma grades and subclasses. INTERPRETATION: We propose that impaired endocytosis creates a selective advantage in glioma tumor progression due to prolonged receptor tyrosine kinase signaling from the cell surface. FUND: This work was supported by Grants 316030-164105 (to P. Jenö), 31003A-162643 (to M. Spiess) and PP00P3-176974 (to G. Hutter) from the Swiss National Science Foundation. Further funding was received by the Department of Surgery from the University Hospital Basel.
Assuntos
Endocitose , Glioma/metabolismo , Proteoma , Proteômica , Biópsia , Biologia Computacional/métodos , Glioma/genética , Glioma/patologia , Humanos , Espectrometria de Massas , Gradação de Tumores , Estadiamento de Neoplasias , Células Neoplásicas Circulantes , Proteômica/métodosRESUMO
Primary aldosteronism is a disease of excessive production of adrenal steroid hormones and the most common cause of endocrine hypertension. Primary aldosteronism results mainly from bilateral adrenal hyperplasia or unilateral aldosterone-producing adenoma (APA). Primary aldosteronism cause at the molecular level is incompletely understood and a targeted treatment preventing excessive adrenal steroid production is not available. Here, we perform deep quantitative proteomic and phosphoproteomic profiling of 6 pairs of APA and adjacent nontumoral adrenal cortex. We show that increased steroidogenesis in APA is accompanied by upregulation of steroidogenic enzymes (HSD3B2, CYP21A2, CYP11B2) and of proteins involved in cholesterol uptake (LSR). We demonstrate that HSD3B2 is phosphorylated at Ser95 or 96 and identify a novel phosphorylation site, Ser489, in CYP21A2, suggesting that steroidogenic enzymes are regulated by phosphorylation. Our analysis also reveals altered ECM (extracellular matrix) composition in APA that affects ECM-cell surface interactions and actin cytoskeleton rearrangements. We show that RHOC, a GTPase controlling actin organization in response to extracellular stimuli, is upregulated in APA and promotes expression of the aldosterone synthase gene CYP11B2. Our data also indicate deregulation of protein N-glycosylation and GABAergic signaling in APAs. Finally, we find that mTORC1 (mammalian target of rapamycin complex 1) signaling is the major pathway deregulated in APA. Our study provides a rich resource for future research on the molecular mechanisms of primary aldosteronism.
Assuntos
Adenoma/metabolismo , Aldosterona/biossíntese , Proteômica/métodos , Matriz Extracelular/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Transdução de Sinais , Ácido gama-Aminobutírico/metabolismoRESUMO
Diffuse gliomas progress by invading neighboring brain tissue to promote postoperative relapse. Transcription factor SOX2 is highly expressed in invasive gliomas and maps to chromosome region 3q26 together with the genes for PI3K/AKT signaling activator PIK3CA and effector molecules of mitochondria fusion and cell invasion, MFN1 and OPA1. Gene copy number analysis at 3q26 from 129 glioma patient biopsies revealed mutually exclusive SOX2 amplifications (26%) and OPA1 losses (19%). Both forced SOX2 expression and OPA1 inactivation increased LN319 glioma cell invasion in vitro and promoted cell dispersion in vivo in xenotransplanted D. rerio embryos. While PI3 kinase activity sustained SOX2 expression, pharmacological PI3K/AKT pathway inhibition decreased invasion and resulted in SOX2 nucleus-to-cytoplasm translocation in an mTORC1-independent manner. Chromatin immunoprecipitation and luciferase reporter gene assays together demonstrated that SOX2 trans-activates PIK3CA and OPA1. Thus, SOX2 activates PI3K/AKT signaling in a positive feedback loop, while OPA1 deletion is interpreted to counteract OPA1 trans-activation. Remarkably, neuroimaging of human gliomas with high SOX2 or low OPA1 genomic imbalances revealed significantly larger necrotic tumor zone volumes, corresponding to higher invasive capacities of tumors, while autologous necrotic cells are capable of inducing higher invasion in SOX2 overexpressing or OPA1 knocked-down relative to parental LN319. We thus propose necrosis volume as a surrogate marker for the assessment of glioma invasive potential. Whereas glioma invasion is activated by a PI3K/AKT-SOX2 loop, it is reduced by a cryptic invasion suppressor SOX2-OPA1 pathway. Thus, PI3K/AKT-SOX2 and mitochondria fission represent connected signaling networks regulating glioma invasion.
Assuntos
Cromossomos Humanos Par 3 , Classe I de Fosfatidilinositol 3-Quinases/genética , GTP Fosfo-Hidrolases/genética , Glioma/genética , Fatores de Transcrição SOXB1/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Variações do Número de Cópias de DNA , GTP Fosfo-Hidrolases/metabolismo , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Humanos , Necrose/genética , Invasividade Neoplásica , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de SinaisRESUMO
Obesity is a major risk factor for insulin resistance and type 2 diabetes. In adipose tissue, obesity-mediated insulin resistance correlates with the accumulation of proinflammatory macrophages and inflammation. However, the causal relationship of these events is unclear. Here, we report that obesity-induced insulin resistance in mice precedes macrophage accumulation and inflammation in adipose tissue. Using a mouse model that combines genetically induced, adipose-specific insulin resistance (mTORC2-knockout) and diet-induced obesity, we found that insulin resistance causes local accumulation of proinflammatory macrophages. Mechanistically, insulin resistance in adipocytes results in production of the chemokine monocyte chemoattractant protein 1 (MCP1), which recruits monocytes and activates proinflammatory macrophages. Finally, insulin resistance (high homeostatic model assessment of insulin resistance [HOMA-IR]) correlated with reduced insulin/mTORC2 signaling and elevated MCP1 production in visceral adipose tissue from obese human subjects. Our findings suggest that insulin resistance in adipose tissue leads to inflammation rather than vice versa.
Assuntos
Resistência à Insulina , Gordura Intra-Abdominal/metabolismo , Macrófagos/metabolismo , Obesidade/metabolismo , Paniculite/metabolismo , Transdução de Sinais , Células 3T3-L1 , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Gordura Intra-Abdominal/patologia , Macrófagos/patologia , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Obesidade/genética , Obesidade/patologia , Paniculite/genética , Paniculite/patologiaRESUMO
BACKGROUND: Uromodulin is the most abundant protein in healthy human urine. Recently it has been suggested as a specific biomarker of renal tubular damage. We have developed a novel pseudo multiple reaction monitoring (pseudo MRM) for the protein's quantification in human urine. RESULTS: Selection of two peptides allowed quantification of uromodulin in human urine. The pseudo MRM quantified uromodulin in healthy individuals between 21 and 1344 nM and in autosomal dominant tubulointerstitial kidney disease-UMOD patients between 2 and 25 nM. CONCLUSION: The pseudo MRM allows greater confidence in assay specificity than traditional MRM methods and quantified uromodulin at concentrations higher than achievable by ELISA. Differences in urinary uromodulin concentration related to the rs4293393 promoter variant in the UMOD gene was confirmed. This method will be used to further investigate uromodulin as a biomarker of renal injury.
Assuntos
Urinálise/métodos , Uromodulina/urina , Adulto , Sequência de Aminoácidos , Biomarcadores/urina , Cromatografia Líquida/métodos , Humanos , Marcação por Isótopo/métodos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Proteólise , Espectrometria de Massas em Tandem/métodos , Uromodulina/análiseRESUMO
Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O2) and releases hydrogen peroxide (H2O2), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys(208)-Cys(241) disulfide in hyperactive Ero1α (Ero1α-C104A/C131A) potentiates H2O2 production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the flavin cofactor where O2 is reduced to H2O2. Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys(208)/Cys(241)-dependent mixed-disulfide complex with Ero1α. The H2O2-detoxifying glutathione peroxidase 8 also binds to the Cys(208)/Cys(241) loop region. Supported by O2 diffusion simulations, these data describe the first enzymatically controlled O2 access into a flavoprotein active site, provide molecular-level understanding of Ero1α regulation and H2O2 production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity.
Assuntos
Apoptose , Dissulfetos/metabolismo , Peróxido de Hidrogênio/metabolismo , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Oxigênio/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Western Blotting , Catálise , Domínio Catalítico , Proliferação de Células , Células Cultivadas , Retículo Endoplasmático , Flavina-Adenina Dinucleotídeo/metabolismo , Imunofluorescência , Células HeLa , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Benzbromarone is an uricosuric structurally related to amiodarone and a known mitochondrial toxicant. The aim of the current study was to improve our understanding in the molecular mechanisms of benzbromarone-associated hepatic mitochondrial toxicity. In HepG2 cells and primary human hepatocytes, ATP levels started to decrease in the presence of 25-50µM benzbromarone for 24-48h, whereas cytotoxicity was observed only at 100µM. In HepG2 cells, benzbromarone decreased the mitochondrial membrane potential starting at 50µM following incubation for 24h. Additionally, in HepG2 cells, 50µM benzbromarone for 24h induced mitochondrial uncoupling,and decreased mitochondrial ATP turnover and maximal respiration. This was accompanied by an increased lactate concentration in the cell culture supernatant, reflecting increased glycolysis as a compensatory mechanism to maintain cellular ATP. Investigation of the electron transport chain revealed a decreased activity of all relevant enzyme complexes. Furthermore, treatment with benzbromarone was associated with increased cellular ROS production, which could be located specifically to mitochondria. In HepG2 cells and in isolated mouse liver mitochondria, benzbromarone also reduced palmitic acid metabolism due to an inhibition of the long-chain acyl CoA synthetase. In HepG2 cells, benzbromarone disrupted the mitochondrial network, leading to mitochondrial fragmentation and a decreased mitochondrial volume per cell. Cell death occurred by both apoptosis and necrosis. The study demonstrates that benzbromarone not only affects the function of mitochondria in HepG2 cells and human hepatocytes, but is also associated with profound changes in mitochondrial structure which may be associated with apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Benzobromarona/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Metabolismo Energético/efeitos dos fármacos , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Glicólise/efeitos dos fármacos , Células Hep G2 , Humanos , Ácido Láctico/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Necrose , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
T cells that recognize self-lipids presented by CD1c are frequent in the peripheral blood of healthy individuals and kill transformed hematopoietic cells, but little is known about their antigen specificity and potential antileukemia effects. We report that CD1c self-reactive T cells recognize a novel class of self-lipids, identified as methyl-lysophosphatidic acids (mLPAs), which are accumulated in leukemia cells. Primary acute myeloid and B cell acute leukemia blasts express CD1 molecules. mLPA-specific T cells efficiently kill CD1c(+) acute leukemia cells, poorly recognize nontransformed CD1c-expressing cells, and protect immunodeficient mice against CD1c(+) human leukemia cells. The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed leukemia control by lipid-specific T cells in vivo provide a new conceptual framework for leukemia immune surveillance and possible immunotherapy.
Assuntos
Antígenos CD1/imunologia , Autoantígenos/imunologia , Crise Blástica/imunologia , Glicoproteínas/imunologia , Vigilância Imunológica , Leucemia Mieloide Aguda/imunologia , Lisofosfolipídeos/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Linfócitos T/imunologia , Adolescente , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Antígenos CD1/genética , Autoantígenos/genética , Crise Blástica/genética , Crise Blástica/patologia , Criança , Pré-Escolar , Feminino , Regulação Leucêmica da Expressão Gênica/genética , Regulação Leucêmica da Expressão Gênica/imunologia , Glicoproteínas/genética , Humanos , Células Jurkat , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Lisofosfolipídeos/genética , Masculino , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Linfócitos T/patologiaRESUMO
A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Antimaláricos/farmacologia , Plasmodium falciparum/fisiologia , Raios Ultravioleta , AnimaisRESUMO
This article presents the major differences in the exoproteomes of Listeria monocytogenes strains grown at 11°C and 20°C, and their comparison to 37°C, the optimal temperature of growth of this foodborne pathogenic bacteria. A set of four strains previously characterized and representing the genetic diversity of the species was used. Two were virulent, of which one was persistent, and two were low virulent strains. The proteins secreted by the strains grown in minimal medium were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by liquid chromatography tandem mass spectrometry. The heterogeneity among the four strains concerning the 15 major proteins detected was noticed. No clear association of exoproteome with virulence or genotype was found. Cluster analysis of the protein patterns of the strains suggests an increasing differentiation of strain response with low temperatures, highlighting the importance of the study of the exoproteomes. The main finding was the lack of some proteins in the exoproteome of the persistent strain, namely, flagellin (FlaA) and of OppA/oligopeptide ABC transporter, when compared to the other strains. In fact, these two proteins differ in abundance between strains grown at low temperature. Moreover, FlaA was the only glycoprotein identified in the exoproteomes. An attempt is made here to assess the relevance of the major exoproteins differentially detected. The investigation of the exoproteomes of other persistent and sporadic strains will allow identification of proteins involved in adaptation of particular L. monocytogenes strains to low temperatures in use throughout the food chain.
Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Listeria monocytogenes/metabolismo , Proteoma , Proteômica/métodos , Proteínas de Transporte/metabolismo , Cromatografia Líquida , Análise por Conglomerados , Temperatura Baixa , Eletroforese em Gel de Poliacrilamida , Flagelina/metabolismo , Lipoproteínas/metabolismo , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/fisiologia , Microscopia Eletrônica de Transmissão , Espectrometria de Massas em Tandem , VirulênciaRESUMO
The evolutionarily conserved Orm1 and Orm2 proteins mediate sphingolipid homeostasis. However, the homologous Orm proteins and the signaling pathways modulating their phosphorylation and function are incompletely characterized. Here we demonstrate that inhibition of nutrient-sensitive target of rapamycin complex 1 (TORC1) stimulates Orm phosphorylation and synthesis of complex sphingolipids in Saccharomyces cerevisiae. TORC1 inhibition activates the kinase Npr1 that directly phosphorylates and activates the Orm proteins. Npr1-phosphorylated Orm1 and Orm2 stimulate de novo synthesis of complex sphingolipids downstream of serine palmitoyltransferase. Complex sphingolipids in turn stimulate plasma membrane localization and activity of the nutrient scavenging general amino acid permease 1. Thus activation of Orm and complex sphingolipid synthesis upon TORC1 inhibition is a physiological response to starvation.
Assuntos
Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/biossíntese , Fatores de Transcrição/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Homeostase , Fosforilação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Fatores de Transcrição/antagonistas & inibidoresRESUMO
A current major obstacle is that no reliable screening markers exist to detect pregnancies at risk for preeclampsia. Quantitative proteomic analysis employing isobaric labelling (iTRAQ) has been suggested to be suitable for the detection of potential plasma biomarkers, a feature we recently verified in analysis of pregnancies with Down syndrome foetuses. We have now examined whether this approach could yield biomarkers to screen pregnancies at risk for preeclampsia. In our study, we used maternal plasma samples obtained at 12 weeks of gestation, six from women who subsequently developed preeclampsia and six with uncomplicated deliveries. In our analysis, we observed elevations in 10 proteins out of 64 proteins in the preeclampsia study group when compared to the healthy control group. These proteins included clusterin, fibrinogen, fibronectin, and angiotensinogen, increased levels of which are known to be associated with preeclampsia. An elevation in the immune-modulatory molecule, galectin 3 binding protein, was also noted. Our pilot study, therefore, indicates that quantitative proteomic iTRAQ analysis could be a useful tool for the detection of new preeclampsia screening markers.
Assuntos
Pré-Eclâmpsia/sangue , Primeiro Trimestre da Gravidez/sangue , Diagnóstico Pré-Natal/métodos , Bases de Dados de Proteínas , Feminino , Humanos , Fragmentos de Peptídeos/sangue , Pré-Eclâmpsia/metabolismo , Gravidez , Proteoma/metabolismo , Proteômica/métodos , Estudos Retrospectivos , Espectrometria de Massas em TandemRESUMO
The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Proteínas de Choque Térmico/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sítios de Ligação , Chlamydomonas reinhardtii/efeitos dos fármacos , Cicloeximida/farmacologia , Chaperona BiP do Retículo Endoplasmático , Glicosilação/efeitos dos fármacos , Resposta ao Choque Térmico , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Treonina , Tunicamicina/farmacologiaRESUMO
Capnocytophaga canimorsus are commensal Gram-negative bacteria from dog's mouth that cause rare but dramatic septicaemia in humans. C. canimorsus have the unusual property to feed on cultured mammalian cells, including phagocytes, by harvesting the glycan moiety of cellular glycoproteins. To understand the mechanism behind this unusual property, the genome of strain Cc5 was sequenced and analysed. In addition, Cc5 bacteria were cultivated onto HEK 293 cells and the surface proteome was determined. The genome was found to encode many lipoproteins encoded within 13 polysaccharide utilization loci (PULs) typical of the Flavobacteria-Bacteroides group. PULs encode surface exposed feeding complexes resembling the archetypal starch utilization system (Sus). The products of at least nine PULs were detected among the surface proteome and eight of them represented more than half of the total peptides detected from the surface proteome. Systematic deletions of the 13 PULs revealed that half of these Sus-like complexes contributed to growth on animal cells. The complex encoded by PUL5, one of the most abundant ones, was involved in foraging glycans from glycoproteins. It was essential for growth on cells and contributed to survival in mice. It thus represents a fitness factor during infection.
Assuntos
Proteínas de Bactérias/metabolismo , Capnocytophaga/metabolismo , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Proteoma/metabolismo , Animais , Linhagem Celular , DNA Bacteriano/química , DNA Bacteriano/genética , Células Epiteliais/microbiologia , Genes Bacterianos , Genoma Bacteriano , Humanos , Redes e Vias Metabólicas/genética , Camundongos , Dados de Sequência Molecular , Família Multigênica , Análise de Sequência de DNARESUMO
C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-ß-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including ß-galactosidases, ß-N-Acetylhexosaminidases and α-mannosidases.
Assuntos
Capnocytophaga/metabolismo , Glicoproteínas/metabolismo , Imunoglobulina G/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Linhagem Celular , Glicosilação , Infecções por Bactérias Gram-Negativas , Humanos , Lipoproteínas/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , alfa-Manosidose/metabolismo , beta-Galactosidase/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Regulation of cell growth requires extensive coordination of several processes including transcription, ribosome biogenesis, translation, nutrient metabolism, and autophagy. In yeast, the protein kinases Target of Rapamycin (TOR) and protein kinase A (PKA) regulate these processes and are thereby the main activators of cell growth in response to nutrients. How TOR, PKA, and their corresponding signaling pathways are coordinated to control the same cellular processes is not understood. Quantitative analysis of the rapamycin-sensitive phosphoproteome combined with targeted analysis of PKA substrates suggests that TOR complex 1 (TORC1) activates PKA but only toward a subset of substrates. Furthermore, we show that TORC1 signaling impinges on BCY1, the negative regulatory subunit of PKA. Inhibition of TORC1 with rapamycin leads to BCY1 phosphorylation on several sites including T129. Phosphorylation of BCY1 T129 results in BCY1 activation and inhibition of PKA. TORC1 inhibits BCY1 T129 phosphorylation by phosphorylating and activating the S6K homolog SCH9 that in turn inhibits the MAP kinase MPK1. MPK1 phosphorylates BCY1 T129 directly. Thus, TORC1 activates PKA toward some substrates by preventing MPK1-mediated activation of BCY1.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Sequência de Aminoácidos , Ativação Enzimática/efeitos dos fármacos , Marcação por Isótopo , Dados de Sequência Molecular , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Currently no specific biomarkers exist for the screening of pregnancies at risk for Down syndrome (DS). Since a quantitative proteomic approach with isobaric labelling (iTRAQ) has recently been suggested to be highly suitable for the discovery of novel plasma biomarkers, we have now used this method to examine for potential quantitative changes in the plasma proteome of the pregnancies bearing DS fetuses in comparison to normal healthy babies. In our study, we used plasma from six women with DS pregnancies and six with uncomplicated pregnancies care were taken to match cases and controls for gestational and maternal age, as these could be a confounder. In our quantitative proteomics analysis we were able to detect 178 proteins using iTRAQ labelling in conjunction with 4800 MALDI TOF/TOF. Amongst these we observed changes in betaHCG, a known screening marker for DS, indicating that our assay was functional. We found a number of elevated proteins Ig lambda chain C region, serum amyloid P-component, amyloid beta A4, and under expressed proteins like gamma-actin and titin in DS pregnancies. These proteins are also found in the sera of patients with Alzheimer disease, which share similar pathologies of DS. Our study therefore indicates that the iTRAQ labelling approach may be indeed useful for the detection of novel biomarkers.
Assuntos
Síndrome de Down/sangue , Síndrome de Down/diagnóstico , Marcação por Isótopo/métodos , Diagnóstico Pré-Natal/métodos , Proteômica/métodos , Adulto , Biomarcadores/sangue , Regulação para Baixo , Feminino , Doenças Fetais/diagnóstico , Humanos , Indicadores e Reagentes , Espectrometria de Massas , Redes e Vias Metabólicas , Gravidez , Proteínas/análise , Proteínas/química , Software , Regulação para CimaRESUMO
Pathogenic mycobacteria survive within macrophages through the inhibition of phagosome-lysosome fusion. A crucial factor for avoiding lysosomal degradation is the mycobacterial serine/threonine protein kinase G (PknG). PknG is released into the macrophage cytosol upon mycobacterial infection, suggesting that PknG might exert its activity by interfering with host signaling cascades, but the mode of action of PknG remains unknown. Here, we show that PknG undergoes autophosphorylation on threonine residues located at the N terminus. In contrast to all other mycobacterial kinases investigated thus far, autophosphorylation of PknG was not involved in the regulation of its kinase activity. However, autophosphorylation was crucial for the capacity of PknG to promote mycobacterial survival within macrophages. These results will contribute to a better understanding of the virulence mechanisms of pathogenic mycobacteria and may help to design improved inhibitors of PknG to be developed as antimycobacterial compounds.