Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 51
Filtrar
1.
Cancers (Basel) ; 14(18)2022 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-36139700

RESUMO

In cancer, the complex interplay between tumor cells and the tumor microenvironment results in the modulation of signaling processes. By assessing the expression of a multitude of proteins and protein variants in cancer tissue, wide-ranging information on signaling pathway activation and the status of the immunological landscape is obtainable and may provide viable information on the treatment response. Archived breast cancer tissues from a cohort of 84 patients (no adjuvant therapy) were analyzed by high-throughput Western blotting, and the expression of 150 proteins covering central cancer pathways and immune cell markers was examined. By assessing CD8α, CD11c, CD16 and CD68 expression, immune cell infiltration was determined and revealed a strong correlation between event-free patient survival and the infiltration of immune cells. The presence of tumor-infiltrating lymphocytes was linked to the pronounced activation of the Jak/Stat signaling pathway and apoptotic processes. The elevated phosphorylation of PPARγ (pS112) in non-immune-infiltrated tumors suggests a novel immune evasion mechanism in breast cancer characterized by increased PPARγ phosphorylation. Multiplexed immune cell marker assessment and the protein profiling of tumor tissue provide functional signaling data facilitating breast cancer patient stratification.

2.
Biol Chem ; 403(3): 331-343, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-34599868

RESUMO

Periportal and perivenous hepatocytes show zonal heterogeneity in metabolism and signaling. Here, hepatic zonation in mouse liver was analyzed by non-targeted mass spectrometry (MS) and by the antibody-based DigiWest technique, yielding a comprehensive overview of protein expression in periportal and perivenous hepatocytes. Targeted immunoaffinity-based proteomics were used to substantiate findings related to drug metabolism. 165 (MS) and 82 (DigiWest) zonated proteins were identified based on the selected criteria for statistical significance, including 7 (MS) and 43 (DigiWest) proteins not identified as zonated before. New zonated proteins especially comprised kinases and phosphatases related to growth factor-dependent signaling, with mainly periportal localization. Moreover, the mainly perivenous zonation of a large panel of cytochrome P450 enzymes was characterized. DigiWest data were shown to complement the MS results, substantially improving possibilities to bioinformatically identify zonated biological processes. Data mining revealed key regulators and pathways preferentially active in either periportal or perivenous hepatocytes, with ß-catenin signaling and nuclear xeno-sensing receptors as the most prominent perivenous regulators, and several kinase- and G-protein-dependent signaling cascades active mainly in periportal hepatocytes. In summary, the present data substantially broaden our knowledge of hepatic zonation in mouse liver at the protein level.


Assuntos
Fígado , Proteômica , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Espectrometria de Massas , Camundongos , Proteínas Quinases/metabolismo
3.
ACS Infect Dis ; 7(6): 1596-1606, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33724771

RESUMO

The presence of antibodies against endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We developed a serum screening platform using a bead-based Western blot system called DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses. Characterization of the immunoassay for detection of SARS-CoV-2 specific antibodies revealed a sensitivity of 90.3% and a diagnostic specificity of 98.1%. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates comparable assay performances (Cohen's κ ranging from 0.8874 to 0.9508). Analogous assays for OC43, 229E, and NL63 were established and combined into one multiplex with the SARS-CoV-2 assay. Seroreactivity for different coronaviruses was detected with high incidence, and the multiplex assay was adapted for serum screening.


Assuntos
COVID-19 , Coronaviridae , Teste para COVID-19 , Humanos , Extratos Vegetais , SARS-CoV-2
4.
mSphere ; 6(1)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627511

RESUMO

The majority of infections with SARS-CoV-2 are asymptomatic or mild without the necessity of hospitalization. It is of importance to reveal if these patients develop an antibody response against SARS-CoV-2 and to define which antibodies confer virus neutralization. We conducted a comprehensive serological survey of 49 patients with a mild course of disease and quantified neutralizing antibody responses against a clinical SARS-CoV-2 isolate employing human cells as targets. Four patients (8%), even though symptomatic, did not develop antibodies against SARS-CoV-2, and two other patients (4%) were positive in only one of the six serological assays employed. For the remaining 88%, antibody response against the S protein correlated with serum neutralization whereas antibodies against the nucleocapsid were poor predictors of virus neutralization. None of the sera enhanced infection of human cells with SARS-CoV-2 at any dilution, arguing against antibody-dependent enhancement of infection in our system. Regarding neutralization, only six patients (12%) could be classified as high neutralizers. Furthermore, sera from several individuals with fairly high antibody levels had only poor neutralizing activity. In addition, employing a novel serological Western blot system to characterize antibody responses against seasonal coronaviruses, we found that antibodies against the seasonal coronavirus 229E might contribute to SARS-CoV-2 neutralization. Altogether, we show that there is a wide breadth of antibody responses against SARS-CoV-2 in patients that differentially correlate with virus neutralization. This highlights the difficulty to define reliable surrogate markers for immunity against SARS-CoV-2.IMPORTANCE There is strong interest in the nature of the neutralizing antibody response against SARS-CoV-2 in infected individuals. For vaccine development, it is especially important which antibodies confer protection against SARS-CoV-2, if there is a phenomenon called antibody-dependent enhancement (ADE) of infection, and if there is cross-protection by antibodies directed against seasonal coronaviruses. We addressed these questions and found in accordance with other studies that neutralization is mediated mainly by antibodies directed against the spike protein of SARS-CoV-2 in general and the receptor binding site in particular. In our test system, utilizing human cells for infection experiments, we did not detect ADE. However, using a novel diagnostic test we found that antibodies against the coronavirus 229E might be involved in cross-protection to SARS-CoV-2.


Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , COVID-19/imunologia , Infecções por Coronavirus/imunologia , SARS-CoV-2/imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Facilitadores/imunologia , Sítios de Ligação/imunologia , Feminino , Hospitalização , Humanos , Masculino , Testes de Neutralização/métodos , Nucleocapsídeo/imunologia , Estações do Ano , Testes Sorológicos/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Inquéritos e Questionários , Vacinas/imunologia
5.
Nat Immunol ; 22(1): 74-85, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32999467

RESUMO

T cell immunity is central for the control of viral infections. To characterize T cell immunity, but also for the development of vaccines, identification of exact viral T cell epitopes is fundamental. Here we identify and characterize multiple dominant and subdominant SARS-CoV-2 HLA class I and HLA-DR peptides as potential T cell epitopes in COVID-19 convalescent and unexposed individuals. SARS-CoV-2-specific peptides enabled detection of post-infectious T cell immunity, even in seronegative convalescent individuals. Cross-reactive SARS-CoV-2 peptides revealed pre-existing T cell responses in 81% of unexposed individuals and validated similarity with common cold coronaviruses, providing a functional basis for heterologous immunity in SARS-CoV-2 infection. Diversity of SARS-CoV-2 T cell responses was associated with mild symptoms of COVID-19, providing evidence that immunity requires recognition of multiple epitopes. Together, the proposed SARS-CoV-2 T cell epitopes enable identification of heterologous and post-infectious T cell immunity and facilitate development of diagnostic, preventive and therapeutic measures for COVID-19.


Assuntos
COVID-19/imunologia , Epitopos de Linfócito T/imunologia , Peptídeos/imunologia , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Vacinas Virais/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Reações Cruzadas/imunologia , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Memória Imunológica/imunologia , SARS-CoV-2/fisiologia , Linfócitos T/metabolismo , Vacinas Virais/administração & dosagem
6.
Nucleic Acids Res ; 48(14): 7748-7766, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32585002

RESUMO

Mouse embryonic stem cells (mESCs) cultured with MEK/ERK and GSK3ß (2i) inhibitors transition to ground state pluripotency. Gene expression changes, redistribution of histone H3K27me3 profiles and global DNA hypomethylation are hallmarks of 2i exposure, but it is unclear whether epigenetic alterations are required to achieve and maintain ground state or occur as an outcome of 2i signal induced changes. Here we show that ESCs with three epitypes, WT, constitutively methylated, or hypomethylated, all undergo comparable morphological, protein expression and transcriptome changes independently of global alterations of DNA methylation levels or changes in H3K27me3 profiles. Dazl and Fkbp6 expression are induced by 2i in all three epitypes, despite exhibiting hypermethylated promoters in constitutively methylated ESCs. We identify a number of activated gene promoters that undergo 2i dependent loss of H3K27me3 in all three epitypes, however genetic and pharmaceutical inhibition experiments show that H3K27me3 is not required for their silencing in non-2i conditions. By separating and defining their contributions, our data suggest that repressive epigenetic systems play minor roles in mESC self-renewal and naïve ground state establishment by core sets of dominant pluripotency associated transcription factor networks, which operate independently from these epigenetic processes.


Assuntos
Repressão Epigenética , Redes Reguladoras de Genes , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Células Cultivadas , Metilação de DNA , Epigênese Genética , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Histonas/metabolismo , Masculino , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/enzimologia , Fatores de Transcrição/metabolismo , Transcrição Gênica
7.
Arch Toxicol ; 94(4): 1265-1278, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32123963

RESUMO

The complex three-dimensional architecture of the liver with its metabolically zonated lobules is a prerequisite to perform functions of metabolic conversion of endogenous and foreign substrates. The enzymatic competencies of hepatocytes differ between zones and dynamically adapt upon xenobiotic activation of the nuclear constitutive androstane receptor (CAR). Using the antibody-based DigiWest proteomics approach, the abundance and phosphorylation status of hepatocyte proteins isolated by laser capture microdissection from the periportal and pericentral regions of murine liver lobules were analyzed. Patterns that distinguish region-specific hepatocytes were detected and the characteristic changes in phosphorylation and phosphatase activity were observed after CAR activation by TCPOBOP in mice. Time- and liver zone-dependent induction of CAR target proteins was monitored. Our observations substantially broaden our knowledge on zone-specific expression and regulation of signaling proteins and metabolic enzymes in different liver zones and their regulation by CAR activation. Inhibition of PP2A was observed in periportal hepatocytes and the amount and phosphorylation state of central hepatic co-regulators such as HNF4α and PGC-1α were altered. Thereby, this analysis of cellular signaling identifies inhibition of PP2A as the central regulatory element governing zonal metabolism. Our study demonstrates the usefulness of the DigiWest approach in unraveling zone-specific hepatic responses to the exposure against xenobiotics.


Assuntos
Fígado/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Western Blotting , Núcleo Celular , Receptor Constitutivo de Androstano , Hepatócitos/metabolismo , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Análise Serial de Proteínas , Piridinas , Transdução de Sinais , Xenobióticos
8.
Mol Cancer Res ; 18(2): 278-286, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31704732

RESUMO

Mechanisms of intrinsic resistance of serous ovarian cancers to standard treatment with carboplatin and paclitaxel are poorly understood. Seventeen primary serous ovarian cancers classified as responders or nonresponders to standard treatment were screened with DigiWest protein array analysis for 279 analytes. Histone methyl transferase EZH2, an interaction partner of ataxia telangiectasia mutated (ATM), was found as one of the most significantly represented proteins in responsive tumors. Survival analysis of 616 patients confirmed a better outcome in patients with high EZH2 expression, but a worse outcome in patients with low EZH2 and high-ATM-expressing tumors compared with patients with low EZH2 and low-ATM-expressing tumors. A proximity ligation assay further confirmed an association between ATM and EZH2 in tumors of patients with an increased disease-free survival. Knockdown of EZH2 resulted in treatment-resistant cells, but suppression of both EZH2 and ATM, or ATM alone, had no effect. DigiWest protein analysis of EZH2-knockdown cells revealed a decrease in proteins involved in mitotic processes and checkpoint regulation, suggesting that deregulated ATM may induce treatment resistance. IMPLICATIONS: Ovarian cancer is a malignancy with high mortality rates, with to date, no successful molecular characterization strategies. Our study uncovers in a comprehensive approach the involvement of checkpoint regulation via ATM and EZH2, potentially providing a new therapeutic perspective for further investigations.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carboplatina/farmacologia , Cistadenocarcinoma Seroso/tratamento farmacológico , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Idoso , Proteínas Mutadas de Ataxia Telangiectasia/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Dano ao DNA , DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos , Proteína Potenciadora do Homólogo 2 de Zeste/deficiência , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
9.
Drug Metab Dispos ; 46(11): 1462-1465, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30115646

RESUMO

Nuclear receptors mediate the hepatic induction of drug-metabolizing enzymes by xenobiotics. Not much is known about enzyme induction in liver tumors. Here, we treated tumor-bearing mice with phenobarbital, an activator of the constitutive androstane receptor (CAR), to analyze the response of chemically induced Ha-ras- and B-raf-mutated mouse liver adenoma to CAR activation in vivo. Both tumor subpopulations possess almost identical gene expression profiles. CAR target gene induction in the tumors was studied at the mRNA and protein levels, and a reverse-phase protein microarray approach was chosen to characterize important signaling cascades. CAR target gene induction was pronounced in B-raf-mutated but not in Ha-ras-mutated tumors. Phosphoproteomic profiling revealed that phosphorylation-activated extracellular signal-regulated kinase (ERK) 1/2 was more abundant in Ha-ras-mutated than in B-raf-mutated tumors. ERK activation in tumor tissue was negatively correlated with CAR target induction. ERK activation is known to inhibit CAR-dependent transcription. In summary, profound differences exist between the two closely related tumor subpopulations with respect to the activation of mitogenic signaling cascades, and these dissimilarities might explain the differences in xenobiotic induction of CAR target genes.


Assuntos
Carcinoma Hepatocelular/genética , Genes ras/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais/genética , Animais , Receptor Constitutivo de Androstano , Neoplasias Hepáticas/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Mutação/efeitos dos fármacos , Fenobarbital/farmacologia , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
10.
Anal Chem ; 90(9): 5788-5794, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29570278

RESUMO

Multitransmembrane proteins are notoriously difficult to analyze. To date, rapid, and cost-efficient detection methods are lacking and only mass spectrometry-based systems allow reliable quantification of these proteins. Here, we present a novel type of sandwich immunoassay that is capable of sensitively detecting multidrug resistance protein 1 (MDR1), a prototypic 12-transmembrane-domains transporter. In a first assay step, complex samples are enzymatically fragmented into peptides as routinely done for mass spectrometry. A proteotypic peptide derived from MDR1 was chosen and antibodies targeting this peptide were used to build a sandwich immunoassay. Validation of the optimized assay showed good sensitivity, reproducibility and it allowed reliable quantification of MDR1; cross-validation by mass spectrometry demonstrated the applicability for routine analyses in clinical and pharmaceutical research. MDR1 was quantified in primary human renal cell carcinoma and corresponding normal tissue and down-regulation or expression loss was found in tumor tissue corroborating its importance in drug resistance and efficacy.


Assuntos
Carcinoma de Células Renais/química , Imunoensaio , Neoplasias Renais/química , Peptídeos/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/análise , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia
11.
Nat Commun ; 7: 12852, 2016 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-27659302

RESUMO

Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes.

12.
J Nat Prod ; 79(4): 1112-23, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-27002382

RESUMO

Impaired wound healing is one of the main risk factors associated with diabetes mellitus. Few options are available to treat diabetic wounds, and therefore efficient remedies are urgently needed. An interesting option might be an extract of birch bark (TE) that has been clinically proven to accelerate acute wound healing. We investigated the effects of TE and its main components betulin and lupeol in cultured normal keratinocytes and dermal fibroblasts from diabetic and nondiabetic donors. These in vitro models can provide insights into possible beneficial effects in wound healing. TE and betulin treatment led to increased mRNA levels of chemokines, pro-inflammatory cytokines, and mediators important in wound healing, e.g., IL-6, TNFα, IL-8, and RANTES. We observed a pronounced upregulation of MIF, IL-8, and RANTES on the protein level. Furthermore, a shape change of the actin cytoskeleton was seen in keratinocytes and fibroblasts, and the Rho-GTPases and p38-MAPK were found to be activated in keratinocytes. On the basis of our results, TE is worthy of further study as a potential option to influence wound-healing processes under diabetic conditions. These first insights need to be confirmed by clinical studies with diabetic patients.


Assuntos
Betula/química , Diabetes Mellitus/tratamento farmacológico , Queratinócitos/efeitos dos fármacos , Triterpenos Pentacíclicos/farmacologia , Triterpenos/isolamento & purificação , Triterpenos/farmacologia , Citocinas/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Triterpenos Pentacíclicos/química , Triterpenos Pentacíclicos/isolamento & purificação , Casca de Planta/química , Triterpenos/química , Cicatrização/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
13.
Arch Toxicol ; 90(6): 1481-94, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26838046

RESUMO

Activation of Wnt/ß-catenin signaling is important for human and rodent hepatocarcinogenesis. In mice, the tumor promoter phenobarbital (PB) selects for hepatocellular tumors with activating ß-catenin mutations via constitutive androstane receptor activation. PB-dependent tumor promotion was studied in mice with genetic inactivation of Apc, a negative regulator of ß-catenin, to circumvent the problem of randomly induced mutations by chemical initiators and to allow monitoring of PB- and Wnt/ß-catenin-dependent tumorigenesis in the absence of unknown genomic alterations. Moreover, the study was designed to investigate PB-induced proliferation of liver cells with activated ß-catenin. PB treatment provided Apc-deficient hepatocytes with only a minor proliferative advantage, and additional connexin 32 deficiency did not affect the proliferative response. PB significantly promoted the outgrowth of Apc-deficient hepatocellular adenoma (HCA), but simultaneously inhibited the formation of Apc-deficient hepatocellular carcinoma (HCC). The probability of tumor promotion by PB was calculated to be much lower for hepatocytes with loss of Apc, as compared to mutational ß-catenin activation. Comprehensive transcriptomic and phosphoproteomic characterization of HCA and HCC revealed molecular details of the two tumor types. HCC were characterized by a loss of differentiated hepatocellular gene expression, enhanced proliferative signaling, and massive over-activation of Wnt/ß-catenin signaling. In conclusion, PB exerts a dual role in liver tumor formation by promoting the growth of HCA but inhibiting the growth of HCC. Data demonstrate that one and the same compound can produce opposite effects on hepatocarcinogenesis, depending on context, highlighting the necessity to develop a more differentiated view on the tumorigenicity of this model compound.


Assuntos
Proteína da Polipose Adenomatosa do Colo/deficiência , Neoplasias Hepáticas Experimentais/induzido quimicamente , Fenobarbital/toxicidade , Transcriptoma/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Proliferação de Células/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Imuno-Histoquímica , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , beta Catenina/genética
15.
PLoS Comput Biol ; 12(1): e1004431, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26727233

RESUMO

During various inflammatory processes circulating cytokines including IL-6, IL-1ß, and TNFα elicit a broad and clinically relevant impairment of hepatic detoxification that is based on the simultaneous downregulation of many drug metabolizing enzymes and transporter genes. To address the question whether a common mechanism is involved we treated human primary hepatocytes with IL-6, the major mediator of the acute phase response in liver, and characterized acute phase and detoxification responses in quantitative gene expression and (phospho-)proteomics data sets. Selective inhibitors were used to disentangle the roles of JAK/STAT, MAPK, and PI3K signaling pathways. A prior knowledge-based fuzzy logic model comprising signal transduction and gene regulation was established and trained with perturbation-derived gene expression data from five hepatocyte donors. Our model suggests a greater role of MAPK/PI3K compared to JAK/STAT with the orphan nuclear receptor RXRα playing a central role in mediating transcriptional downregulation. Validation experiments revealed a striking similarity of RXRα gene silencing versus IL-6 induced negative gene regulation (rs = 0.79; P<0.0001). These results concur with RXRα functioning as obligatory heterodimerization partner for several nuclear receptors that regulate drug and lipid metabolism.


Assuntos
Hepatócitos/metabolismo , Inativação Metabólica/fisiologia , Inflamação/metabolismo , Modelos Biológicos , Receptor X Retinoide alfa/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biologia Computacional , Regulação para Baixo , Feminino , Lógica Fuzzy , Humanos , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , Adulto Jovem
16.
Gut ; 65(5): 840-51, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-25652085

RESUMO

OBJECTIVE: Alcoholic hepatitis (AH) is often associated with advanced fibrosis, which negatively impacts survival. We aimed at identifying kinases deregulated in livers from patients with AH and advanced fibrosis in order to discover novel molecular targets. DESIGN: Extensive phosphoprotein analysis by reverse phase protein microarrays was performed in AH (n=12) and normal human livers (n=7). Ribosomal S6 kinase (p90RSK) hepatic expression was assessed by qPCR, Western blot and immunohistochemistry. Kaempferol was used as a selective pharmacological inhibitor of the p90RSK pathway to assess the regulation of experimentally-induced liver fibrosis and injury, using in vivo and in vitro approaches. RESULTS: Proteomic analysis identified p90RSK as one of the most deregulated kinases in AH. Hepatic p90RSK gene and protein expression was also upregulated in livers with chronic liver disease. Immunohistochemistry studies showed increased p90RSK staining in areas of active fibrogenesis in cirrhotic livers. Therapeutic administration of kaempferol to carbon tetrachloride-treated mice resulted in decreased hepatic collagen deposition, and expression of profibrogenic and proinflammatory genes, compared to vehicle administration. In addition, kaempferol reduced the extent of hepatocellular injury and degree of apoptosis. In primary hepatic stellate cells, kaempferol and small interfering RNA decreased activation of p90RSK, which in turn regulated key profibrogenic actions. In primary hepatocytes, kaempferol attenuated proapoptotic signalling. CONCLUSIONS: p90RSK is upregulated in patients with chronic liver disease and mediates liver fibrogenesis in vivo and in vitro. These results suggest that the p90RSK pathway could be a new therapeutic approach for liver diseases characterised by advanced fibrosis.


Assuntos
Hepatite Alcoólica/complicações , Hepatite Alcoólica/enzimologia , Cirrose Hepática/etiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade
17.
PLoS One ; 10(12): e0144535, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26695635

RESUMO

Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.


Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos de Superfície/imunologia , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Células Neoplásicas Circulantes/metabolismo , Adulto , Idoso , Antígenos de Neoplasias/sangue , Antígenos de Superfície/sangue , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/sangue , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Molécula de Adesão da Célula Epitelial , Proteínas da Matriz Extracelular/sangue , Feminino , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Metástase Neoplásica , Análise de Célula Única
18.
Methods Mol Biol ; 1348: 251-65, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26424278

RESUMO

Modification state-specific antibodies are powerful tools for investigating posttranslational modifications in proteins. The majority of these antibodies have been generated against peptide-antigen conjugates. They are useful in a plethora of methods, such as Western blotting, immunohistochemistry, sandwich immunoassay, immunoprecipitation, and immunoprecipitation coupled with mass spectrometry. Phosphorylation, acetylation, methylation, sulfation, nitrosylation, ubiquitination, and sumoylation are some of the modifications that can be studied using such antibodies. However, investigating the on- and off-target binding of antibodies is crucial to the interpretation of experimental data. Peptide arrays are excellent tools for such in-depth studies of off-target and on-target binding of antibodies. Dozens or even hundreds of modified peptides can be integrated into a single experimental setup to analyze the antibody's binding behavior. Here, we propose three different protocols for peptide bead array generation and describe their suitability for such types of assay.


Assuntos
Anticorpos/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Peptídeos/imunologia , Análise Serial de Proteínas/métodos , Especificidade de Anticorpos/imunologia , Epitopos/química , Peptídeos/química
19.
Sci Rep ; 5: 8759, 2015 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-25737130

RESUMO

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system.


Assuntos
Hepatócitos/enzimologia , Hepatócitos/metabolismo , Espectrometria de Massas/métodos , Peptídeos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/imunologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Anticorpos/imunologia , Afinidade de Anticorpos , Hidrocarboneto de Aril Hidroxilases/imunologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Atorvastatina/farmacocinética , Células Cultivadas , Cromatografia Líquida/métodos , Citocromo P-450 CYP3A/imunologia , Citocromo P-450 CYP3A/metabolismo , Epitopos/imunologia , Epitopos/metabolismo , Hepatócitos/citologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Peptídeos/imunologia , Pravastatina/farmacocinética , Cultura Primária de Células , Homologia de Sequência de Aminoácidos
20.
Biosystems ; 122: 19-24, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24951946

RESUMO

BACKGROUND AND SCOPE: Today, web-based data analysis pipelines exist for a wide variety of microarray platforms, such as ordinary gene-centered arrays, exon arrays and SNP arrays. However, most of the available software tools provide only limited support for reverse-phase protein arrays (RPPA), as relevant inherent properties of the corresponding datasets are not taken into account. Thus, we developed the web-based data analysis pipeline RPPApipe, which was specifically tailored to suit the characteristics of the RPPA platform and encompasses various tools for data preprocessing, statistical analysis, clustering and pathway analysis. IMPLEMENTATION AND PERFORMANCE: All tools which are part of the RPPApipe software were implemented using R/Bioconductor. The software was embedded into our web-based ZBIT Bioinformatics Toolbox which is a customized instance of the Galaxy platform. AVAILABILITY: RPPApipe is freely available under GNU Public License from http://webservices.cs.uni-tuebingen.de. A full documentation of the tool can be found on the corresponding website http://www.cogsys.cs.uni-tuebingen.de/software/RPPApipe.


Assuntos
Biologia Computacional/métodos , Interpretação Estatística de Dados , Análise Serial de Proteínas/métodos , Software , Internet
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA