RESUMO
In mass spectrometry-based proteomics, heavy internal standards are used to validate target peptide detections and to calibrate peptide quantitation. Here, we report light contamination present in heavy labelled synthetic peptides of high isotopic enrichment. Application of such peptides as assay-internal standards potentially compromises the detection and quantitation especially of low abundant cellular peptides. Therefore, it is important to adopt guidelines to prevent false-positive identifications of endogenous light peptides as well as errors in their quantitation from biological samples.
Assuntos
Peptídeos , Proteômica , Marcação por Isótopo , Isótopos , Espectrometria de Massas , Padrões de ReferênciaRESUMO
Summary: Mass spectrometry-based proteomics is increasingly employed in biology and medicine. To generate reliable information from large datasets and ensure comparability of results, it is crucial to implement and standardize the quality control of the raw data, the data processing steps and the statistical analyses. MSPypeline provides a platform for importing MaxQuant output tables, generating quality control reports, data preprocessing including normalization and performing exploratory analyses by statistical inference plots. These standardized steps assess data quality, provide customizable figures and enable the identification of differentially expressed proteins to reach biologically relevant conclusions. Availability and implementation: The source code is available under the MIT license at https://github.com/siheming/mspypeline with documentation at https://mspypeline.readthedocs.io. Benchmark mass spectrometry data are available on ProteomeXchange (PXD025792). Supplementary information: Supplementary data are available at Bioinformatics Advances online.
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The IL-1ß induced activation of the p38MAPK/MAPK-activated protein kinase 2 (MK2) pathway in hepatocytes is important for control of the acute phase response and regulation of liver regeneration. Many aspects of the regulatory relevance of this pathway have been investigated in immune cells in the context of inflammation. However, very little is known about concentration-dependent activation kinetics and signal propagation in hepatocytes and the role of MK2. We established a mathematical model for IL-1ß-induced activation of the p38MAPK/MK2 pathway in hepatocytes that was calibrated to quantitative data on time- and IL-1ß concentration-dependent phosphorylation of p38MAPK and MK2 in primary mouse hepatocytes. This analysis showed that, in hepatocytes, signal transduction from IL-1ß via p38MAPK to MK2 is characterized by strong signal amplification. Quantification of p38MAPK and MK2 revealed that, in hepatocytes, at maximum, 11.3% of p38MAPK molecules and 36.5% of MK2 molecules are activated in response to IL-1ß. The mathematical model was experimentally validated by employing phosphatase inhibitors and the p38MAPK inhibitor SB203580. Model simulations predicted an IC50 of 1-1.2 µm for SB203580 in hepatocytes. In silico analyses and experimental validation demonstrated that the kinase activity of p38MAPK determines signal amplitude, whereas phosphatase activity affects both signal amplitude and duration. p38MAPK and MK2 concentrations and responsiveness toward IL-1ß were quantitatively compared between hepatocytes and macrophages. In macrophages, the absolute p38MAPK and MK2 concentration was significantly higher. Finally, in line with experimental observations, the mathematical model predicted a significantly higher half-maximal effective concentration for IL-1ß-induced pathway activation in macrophages compared with hepatocytes, underscoring the importance of cell type-specific differences in pathway regulation.
Assuntos
Hepatócitos/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Biológicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Hepatócitos/citologia , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Strong, sequence-specific gas-phase bindings between proline-rich peptides and alkaline earth metal ions in nanoESI-MS experiments were reported by Lehmann et al. (Rapid Commun. Mass Spectrom. 2006, 20, 2404-2410), however its relevance for physiological-like aqueous phase is uncertain. Therefore, the complexes should also be studied in aqueous solution and the relevance of the MS method for binding studies be evaluated. A mobility shift ACE method was used for determining the binding between the small peptide GAPAGPLIVPY and various metal ions in aqueous solution. The findings were compared to the MS results and further explained using computational methods. While the MS data showed a strong alkaline earth ion binding, the ACE results showed nonsignificant binding. The proposed vacuum state complex also decomposed during a molecular dynamic simulation in aqueous solution. This study shows that the formed stable peptide-metal ion adducts in the gas phase by ESI-MS does not imply the existence of analogous adducts in the aqueous phase. Comparing peptide-metal ion interaction under the gaseous MS and aqueous ACE conditions showed huge difference in binding behavior.
Assuntos
Cálcio/química , Eletroforese Capilar/métodos , Gases/química , Peptídeos/química , Espectrometria de Massas , Simulação de Dinâmica MolecularRESUMO
Phosphorylation is known to have a strong impact on protein functions. We analyzed members of the lectin family of multifunctional galectins as targets of the protein kinases CK1, CK2, and PKA. Galectins are potent growth regulators able to bind both glycan and peptide motifs at intra- and extracellular sites. Performing in vitro kinase assays, galectin phosphorylation was detected by phosphoprotein staining and autoradiography. The insertion of phosphoryl groups varied to a large extent depending on the type of kinase applied and the respective galectin substrate. Sites of phosphorylation observed in the recombinant galectins were determined by a strategic combination of phosphopeptide enrichment and nano-ultra-performance liquid chromatography tandem mass spectrometry (nanoUPLC-MS/MS). By in silico modeling, phosphorylation sites were visualized three-dimensionally. Our results reveal galectin-type-specific Ser-/Thr-dependent phosphorylation beyond the known example of galectin-3. These data are the basis for functional studies and also illustrate the analytical sensitivity of the applied methods for further work on human lectins.
Assuntos
Caseína Quinase II/metabolismo , Caseína Quinase I/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Galectinas/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Cromatografia Líquida de Alta Pressão , Galectinas/química , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em TandemRESUMO
AIM: To analyze phospholipid profiles in intrahepatic bile from patients with primary sclerosing cholangitis (PSC) and secondary sclerosing cholangitis (SSC). METHODS: Intrahepatic bile specimens collected via endoscopic retrograde cholangiography from 41 patients were analyzed. Fourteen of these patients were diagnosed with PSC, 10 with SSC, 11 with choledocholithiasis or no identifiable biliary disease, and 6 with cholangiocellular carcinoma (CCC). Bile acid, cholesterol, protein, and bilirubin contents as well as pancreas lipase activity in bile were determined by biochemical methods. Phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) species were quantified using nano-electrospray ionization tandem mass spectrometry. RESULTS: Bile from all the examined patient groups showed a remarkably similar PC and LPC species composition, with only minor statistical differences. Total biliary PC concentrations were highest in controls (8030 ± 1843 µmol/L) and lowest in patients with CCC (1969 ± 981 µmol/L) (P = 0.005, controls vs SSC and CCC, respectively, P < 0.05). LPC contents in bile were overall low (4.2% ± 1.8%). Biliary LPC/PC ratios and ratios of biliary PC to bilirubin, PC to cholesterol, PC to protein, and PC to bile acids showed no intergroup differences. CONCLUSION: PC and LPC profiles being similar in patients with or without sclerosing cholangitis, these phospholipids are likely not of major pathogenetic importance in this disease group.
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Bile/metabolismo , Colangite Esclerosante/metabolismo , Lisofosfatidilcolinas/metabolismo , Fosfatidilcolinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Bile/microbiologia , Estudos de Casos e Controles , Colangite Esclerosante/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
Malaria causes millions of death cases per year. Since Plasmodium falciparum rapidly develops drug resistance, it is of high importance to investigate potential drug targets which may lead to novel rational therapy approaches. Here we report on the interaction of translationally controlled tumor protein of P. falciparum (PfTCTP) with the anti-malarial drug artemisinin. Furthermore, we investigated the crystal structure of PfTCTP. Using mass spectrometry, bioinformatic approaches and surface plasmon resonance spectroscopy, we identified novel binding sites of artemisinin which are in direct neighborhood to amino acids 19-46, 108-134 and 140-163. The regions covered by these residues are known to be functionally important for TCTP function. We conclude that interaction of artemisinin with TCTP may be at least in part explain the antimalarial activity of artemisinin.
Assuntos
Antimaláricos/química , Artemisininas/química , Biomarcadores Tumorais/química , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/química , Sítios de Ligação , Biomarcadores Tumorais/metabolismo , Simulação por Computador , Cristalografia por Raios X , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Ressonância de Plasmônio de Superfície , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
In most solid cancers, cells harboring oncogenic mutations represent only a sub-fraction of the entire population. Within this sub-fraction the expression level of mutated proteins can vary significantly due to cellular variability limiting the efficiency of targeted therapy. To address the causes of the heterogeneity, we performed a systematic analysis of one of the most frequently mutated pathways in cancer cells, the phosphatidylinositol 3 kinase (PI3K) signaling pathway. Among others PI3K signaling is activated by the hepatocyte growth factor (HGF) that regulates proliferation of hepatocytes during liver regeneration but also fosters tumor cell proliferation. HGF-mediated responses of PI3K signaling were monitored both at the single cell and cell population level in primary mouse hepatocytes and in the hepatoma cell line Hepa1_6. Interestingly, we observed that the HGF-mediated AKT responses at the level of individual cells is rather heterogeneous. However, the overall average behavior of the single cells strongly resembled the dynamics of AKT activation determined at the cell population level. To gain insights into the molecular cause for the observed heterogeneous behavior of individual cells, we employed dynamic mathematical modeling in a stochastic framework. Our analysis demonstrated that intrinsic noise was not sufficient to explain the observed kinetic behavior, but rather the importance of extrinsic noise has to be considered. Thus, distinct from gene expression in the examined signaling pathway fluctuations of the reaction rates has only a minor impact whereas variability in the concentration of the various signaling components even in a clonal cell population is a key determinant for the kinetic behavior.
RESUMO
Reversible protein phosphorylation is a key mediator for intracellular signal transduction. Here we report an innovative method for accurate, site-specific protein phosphorylation degree determination by nanoLC-ESI-MS/MS. A stable isotope-labeled pair of peptide/phosphopeptide standards with volumetrically defined molar ratio is used as reference, providing an internal standard for both the analyte peptide and the phosphopeptide. For the preparation of one-source peptide/phosphopeptide standards, an aliquot of the labeled phosphopeptide standard is quantitatively dephosphorylated, yielding an equimolar solution of the peptide standard. Subsequently, the two solutions are mixed at a 1:1 or other volumetric ratio, which equals the molar ratio. This procedure assures a defined concentration ratio of both components that is independent from their absolute concentration. We demonstrate the applicability of the one-source peptide/phosphopeptide standard method by determining the phosphorylation degree of the signalling proteins STAT5A/B and STAT6.
Assuntos
Doença de Hodgkin/metabolismo , Fosfopeptídeos/análise , Proteômica , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição STAT6/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Cromatografia Líquida , Doença de Hodgkin/patologia , Humanos , Fosforilação , Padrões de Referência , Espectrometria de Massas por Ionização por Electrospray , Células Tumorais CultivadasRESUMO
Primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) share a frequent constitutive activation of JAK (Janus kinase)/STAT signaling pathway. Because of complex, nonlinear relations within the pathway, key dynamic properties remained to be identified to predict possible strategies for intervention. We report the development of dynamic pathway models based on quantitative data collected on signaling components of JAK/STAT pathway in two lymphoma-derived cell lines, MedB-1 and L1236, representative of PMBL and cHL, respectively. We show that the amounts of STAT5 and STAT6 are higher whereas those of SHP1 are lower in the two lymphoma cell lines than in normal B cells. Distinctively, L1236 cells harbor more JAK2 and less SHP1 molecules per cell than MedB-1 or control cells. In both lymphoma cell lines, we observe interleukin-13 (IL13)-induced activation of IL4 receptor α, JAK2, and STAT5, but not of STAT6. Genome-wide, 11 early and 16 sustained genes are upregulated by IL13 in both lymphoma cell lines. Specifically, the known STAT-inducible negative regulators CISH and SOCS3 are upregulated within 2 hours in MedB-1 but not in L1236 cells. On the basis of this detailed quantitative information, we established two mathematical models, MedB-1 and L1236 model, able to describe the respective experimental data. Most of the model parameters are identifiable and therefore the models are predictive. Sensitivity analysis of the model identifies six possible therapeutic targets able to reduce gene expression levels in L1236 cells and three in MedB-1. We experimentally confirm reduction in target gene expression in response to inhibition of STAT5 phosphorylation, thereby validating one of the predicted targets.
Assuntos
Doença de Hodgkin/metabolismo , Interleucina-13/farmacologia , Linfoma de Células B/metabolismo , Neoplasias do Mediastino/metabolismo , Modelos Biológicos , Terapia de Alvo Molecular/métodos , Linhagem Celular Tumoral , Doença de Hodgkin/genética , Doença de Hodgkin/terapia , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/terapia , Neoplasias do Mediastino/genética , Neoplasias do Mediastino/terapia , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
BACKGROUND: Phospholipids are essential for the normal function of the intestinal mucus barrier. The objective of this study was to systematically investigate phospholipids in the intestinal mucus of humans suffering from inflammatory bowel diseases, where a barrier defect is strongly supposed to be pathogenetic. METHODS: Optimal mucus recovery was first validated in healthy mice and the method was then transferred to the endoscopic acquisition of ileal and colonic mucus from 21 patients with ulcerative colitis (UC), 10 patients with Crohn's disease (CD), and 29 healthy controls. Nano-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to determine phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) in lipid extracts of mucus specimens. RESULTS: Human and rodent mucus contained very similar phospholipid species. In the ileal and colonic mucus from patients suffering from UC, the concentration of PC was highly significantly lower (607 +/- 147 pmol/100 microg protein and 745 +/- 148 pmol/100 microg protein) compared to that of patients with CD (3223 +/- 1519 pmol/100 microg protein and 2450 +/- 431 pmol/100 microg protein) and to controls (3870 +/- 760 pmol/100 microg protein and 2790 +/- 354 pmol/100 microg protein); overall, P = 0.0002 for ileal specimens and P < 0.0001 for colonic specimens. Independent of disease activity, patients suffering from UC showed an increased saturation grade of PC fatty acid residues and a higher LPC-to-PC ratio. CONCLUSIONS: The intestinal mucus barrier of patients with UC is significantly altered concerning its phospholipid concentration and species composition. These alterations may be very important for the pathogenesis of this disease and underline new therapeutic strategies.
Assuntos
Colite Ulcerativa/etiologia , Colite Ulcerativa/patologia , Mucosa Intestinal/patologia , Muco/metabolismo , Fosfolipídeos/metabolismo , Corticosteroides/uso terapêutico , Animais , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Colo/metabolismo , Colo/patologia , Colonoscopia , Feminino , Humanos , Íleo/metabolismo , Íleo/patologia , Mucosa Intestinal/metabolismo , Lisofosfatidilcolinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Pessoa de Meia-Idade , Fosfatidilcolinas/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Esfingomielinas/metabolismoRESUMO
Matrix-assisted laser desorption ionization (MALDI) has become an enabling technology for the fields of protein mass spectrometry (MS) and proteomics. Despite its widespread use, for example, in protein identification via peptide mass fingerprinting, a comprehensive model for the generation of free gas-phase ions has not yet been developed. All matrices in use today, such as alpha-cyano-4-hydroxycinnamic acid (CHCA), have been found empirically and stem from the early days of MALDI. By systematic and targeted variation of the functional groups of the alpha-cyanocinnamic acid core unit, 4-chloro-alpha-cyanocinnamic acid (Cl-CCA) was selected and synthesized, and it exhibited outstanding matrix properties. Key features are a substantial increase in sensitivity and a considerably enhanced peptide recovery in proteomic analyses because of a much more uniform response to peptides of different basicity. Using Cl-CCA as a matrix for a 1 fmol bovine serum albumin (BSA) in-solution digest, the sequence coverage is raised to 48%, compared with 4% for CHCA. For a gel band containing 25 fmol of BSA, unambiguous protein identification becomes possible with Cl-CCA. These findings also imply ion formation via a chemical ionization mechanism with proton transfer from a reactive protonated matrix species to the peptide analytes. The considerable increase in performance promises to have a strong impact on future analytical applications of MALDI, because current sensitivity limits are overcome and more comprehensive analyses come into reach.
Assuntos
Cinamatos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Peptídeos/análise , Proteômica/métodos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normasRESUMO
Galectin-3 has a unique modular design. Its short N-terminal stretch can be phosphorylated, relevant for nuclear export and anti-anoikis/apoptosis activity. Enzymatic modification by casein kinase 1 at constant ATP concentration yielded mg quantities of mono- and diphosphorylated derivatives at Ser5/Ser11 in a 2:1 ratio. Their carbohydrate-inhibitable binding to asialofetuin, cell surfaces of three tumor lines, rabbit erythrocytes leading to haemagglutination and cytoplasmic sites in fixed tissue sections was not markedly altered relative to phosphate-free galectin-3. Spectroscopically, phosphorylation induced alterations in the far UV CD, indicative of an increase in ordered structure. This is accompanied by changes in the environment of aromatic amino acids signified by shifts in the near UV CD.
Assuntos
Galectina 3/química , Galectina 3/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Assialoglicoproteínas/metabolismo , Caseína Quinase I/metabolismo , Linhagem Celular Tumoral , Dicroísmo Circular , Eritrócitos/citologia , Eritrócitos/metabolismo , Fetuínas , Citometria de Fluxo , Galectina 3/genética , Humanos , Jejuno/metabolismo , Rim/metabolismo , Lactose/metabolismo , Lactose/farmacologia , Camundongos , Dados de Sequência Molecular , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Coelhos , Serina/metabolismo , Espectrofotometria Ultravioleta , alfa-Fetoproteínas/metabolismoRESUMO
Silybin or silymarin extract has been used to treat liver diseases, and has now been entered into clinical trials for cancer treatment. Here, we compared antioxidant and anticancer activities between silybin and its oxidized form 2,3-dehydrosilybin (DHS). With IC50 at three-fold lower concentrations than silybin, DHS inhibited reactive oxygen species generation in glucose-glucose oxidase system and HepG2 cells. Compared with silybin, DHS elicited greater protection against H2O2-induced HepG2 cell death and galactosamine-induced liver injury in vivo. It is known that oxidants induce releases of metalloproteinases (MMP)-2,-9 which are responsible for invasive and metastasis potentials of transformed cells. DHS at 10 microM markedly inhibited MMP-2,-9 releases as well as invasiveness, while silybin at 90 microM had marginal effects. DHS but not silybin at 30 microM induced apoptosis and loss of mitochondrial membrane potentials. LD50 of DHS was five-fold lower than that of silybin. Our data suggest that DHS may be more useful therapeutically than silybin.
Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Animais , Antineoplásicos/química , Antioxidantes/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas , Relação Dose-Resposta a Droga , Galactosamina , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Hepatopatias/metabolismo , Hepatopatias/prevenção & controle , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Espécies Reativas de Oxigênio/metabolismo , Silibina , Silimarina/química , Silimarina/farmacologia , Silimarina/uso terapêuticoRESUMO
Proteins, such as the raf kinase inhibitory protein (RKIP), serve as modulators of signalling pathways by either promoting or inhibiting the formation of productive signalling complexes through protein-protein interactions. In the present study, the plasmodial RKIP ortholog, PfPE-PB1, was cloned, recombinantly expressed and purified to homogeneity. The purified protein was used to investigate the effect of plasmodial RKIP on the autophosphorylation and substrate phosphorylation activity of Plasmodium falciparum calcium-dependent protein kinase 1, PfCDPK1. Phosphorylation of RKIP by PfCDPK1 in in vitro kinase assays suggests that RKIP may be an in vivo substrate of this kinase, although the specific activity of PfCDPK1 is approximately seven-fold lower when RKIP, instead of casein, an exogenous substrate of this enzyme, is used as a substrate. In addition to the observed phosphorylation of RKIP itself, its presence in the assays greatly enhanced the autophosphorylation capacity of PfCDPK1 by approximately 5.5-fold. This substantial increase in autophosphorylation activity was associated with a diminished substrate phosphorylation activity of PfCDPK1 when casein was used. At the same time, RKIP phosphorylation slightly increased when casein was included into the assays. Thus, RKIP is recognized as a substrate under in vitro conditions and appears to act as a regulator of PfCDPK1 kinase activity, which possibly is one of its actual functions in the parasite.
Assuntos
Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Plasmodium falciparum/enzimologia , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Proteína de Ligação a Fosfatidiletanolamina/genética , Fosforilação , Fosfosserina/metabolismo , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Transcrição Gênica/genéticaRESUMO
Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.
Assuntos
Conexina 43/metabolismo , Isoformas de Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Conexina 43/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Dados de Sequência Molecular , Ubiquitina-Proteína Ligases Nedd4 , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Membrane proteins differentially expressed in human papillomavirus type 16 (HPV-16) E5-transfected HaCaT cells have been identified. Membrane proteins were isolated and separated by two-dimensional gel electrophoresis. Spots showing quantitative differences between E5-transfected and control cells were extracted and the proteins were identified by nanoelectrospray ionization mass spectrometry. A total of 24 spots was analysed. Among the proteins showing differential expression, a decreased amount of calnexin and increased expression of hsp70, proteins both involved in maturation and transport of MHC class I complexes to the plasma membrane, were noticed. These findings correlate with the decreased surface expression of MHC class I molecules described in E5-expressing cells, HPV-positive cervical lesions and cervical carcinomas. These results stress the value of the proteomic approach, as used here in the experimental design, which allows the correlation of changes in host gene expression with biological functions of viral genes.
Assuntos
Queratinócitos/química , Proteínas de Membrana/análise , Proteínas Oncogênicas Virais/fisiologia , Antígenos de Histocompatibilidade/metabolismo , Humanos , Queratinócitos/virologia , Espectrometria de Massas por Ionização por Electrospray , TransfecçãoRESUMO
BACKGROUND: Intestinal mucus not only facilitates substrate absorption, but also forms a hydrophobic, phosphatidylcholine (PC) enriched, barrier against luminal gut contents. METHODS: For evaluation of the origin of PC in intestinal mucus, we first analyzed the mucus PC in mice with absent biliary phospholipid secretion (mdr2 (-/-) mice) using electrospray ionization (ESI) tandem mass spectroscopy (MS/MS). Second, in situ perfused rat jejunum, ileum and colon were analyzed after i.v. bolus injections of 155 pmol [(3)H]-PC. Additional in vitro experiments were performed with isolated mucosal cells after incubation with the PC precursor [(3)H]-choline. RESULTS: In mdr2 (-/-) mice and control animals no significant quantitative difference in mucus PC was found, indicating that mucus PC is of intestinal and not biliary origin. In situ perfusion studies detected intestinal secretion of [(3)H]-PC, which was stimulated in presence of 2 mM taurocholate (TC). Secretion rates of [(3)H]-PC were highest in ileum (9.0+/-0.8 fmol h(-1)xcm(-1)), lower in jejunum (4.3+/-0.5) and minimal in colon (0.8+/-0.2). It compares to an intestinal secretion of native PC originating to 64% from bile, 9% from jejunum, 28% from ileum, and 1% from colon. Complementary in vitro studies showed 30-min secretion rates for [(3)H]-PC to be highest in enterocytes from ileum (26.5+/-5.3% of intracellular [(3)H]-PC) and jejunum (19.8+/-2.9%), and significantly lower in colonocytes (8.4+/-1.3%). CONCLUSION: PC in the intestinal mucus originates from secretion by ileal and jejunal enterocytes.
Assuntos
Íleo/metabolismo , Fosfatidilcolinas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Colo/metabolismo , Células Epiteliais/metabolismo , Jejuno/metabolismo , Masculino , Camundongos , Muco/metabolismo , Ratos , Trítio/metabolismoRESUMO
Leukotrienes (LTs) are active lipid mediators derived in the 5-lipoxygenase pathway. LTC(4), the primary cysteinyl LT, is cleaved by gamma-glutamyl transpeptidase (GGT), resulting in LTD(4). We studied the synthesis and metabolism of LTs in three patients with GGT deficiency. LTs were analyzed in urine, plasma, and monocytes after HPLC separation by enzyme immunoassays, radioactivity detection, and electrospray tandem mass spectrometry. Analysis of LTs in urine revealed increased concentrations of LTC(4) (12.8-17.9 nmol/mol creatinine; controls, <0.005 nmol/mol creatinine), whereas LTE(4) was below the detection limit (<0.005 nmol/mol creatinine; controls, 32.2 +/- 8.6 nmol/mol creatinine). In plasma of one patient, LTC(4) was found to be increased (17.3 ng/ml; controls, 9.6 +/- 0.4 ng/ml), whereas LTD(4) and LTE(4) were below the detection limit (<0.005 ng/ml). LTB(4) was found within normal ranges. In contrast to controls, the synthesis of LTD(4) and LTE(4) in stimulated monocytes was below the detection limit (<0.1 ng/10(6) cells; controls, 37.1 +/- 4.8 cells and 39.4 +/- 5.6 ng/10(6) cells, respectively). The formation of [(3)H]LTD(4) from [(3)H]LTC(4) in monocytes was completely deficient (<0.1%; controls, 85 +/- 7%). Our data demonstrate a complete deficiency of LTD(4) biosynthesis in patients with a genetic deficiency of GGT. GGT deficiency represents a new inborn error of cysteinyl LT synthesis and provides a unique model in which to study the pathobiological coherence of LT and glutathione metabolism.
Assuntos
Leucotrienos/metabolismo , gama-Glutamiltransferase/deficiência , Adulto , Glutationa/sangue , Humanos , Leucotrieno C4/biossíntese , Leucotrieno C4/sangue , Leucotrieno C4/urina , Leucotrieno D4/biossíntese , Leucotrieno D4/sangue , Leucotrieno D4/urina , Leucotrienos/sangue , Leucotrienos/urina , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Trítio , gama-Glutamiltransferase/genéticaRESUMO
Using the combination of in-gel elastase digestion, immobilized metal affinity chromatography and high resolution electrospray tandem mass spectrometry, the phosphorylation sites of two phosphoproteins were determined. Complete coverage of all phosphorylation sites (Ser10, Ser139, Thr197, Ser338) of the model phosphoprotein protein kinase A C(alpha)-subunit could be achieved by this strategy in the low picomole range. In addition, three previously unknown phosphorylation sites of the human transcription initiation factor TIF-IA (Ser44, Ser170, Ser172) were determined in this way. Both phosphoproteins could be identified in a protein database on the basis of their elastase generated phosphopeptides alone. The data of seven phosphopeptides were used for identification of protein kinase A, and those of two phosphopeptides for TIF-IA, respectively. The accurate mass data of the electrospray mass spectra recorded at high resolution are extremely useful for sequencing of the elastase generated phosphopeptides and for protein identification by database searching.