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1.
Virchows Arch ; 476(1): 3-15, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31701221

RESUMO

Although traditional morphological evaluation remains the cornerstone for the diagnosis of soft tissue tumors, ancillary diagnostic modalities such as immunohistochemistry and molecular genetic analysis are of ever-increasing importance in this field. New insights into the molecular pathogenesis of soft tissue tumors, often obtained from high-throughput sequencing technologies, has enabled significant progress in the characterization and biologic stratification of mesenchymal neoplasms, expanding the spectrum of immunohistochemical tests (often aimed towards recently discovered genetic events) and molecular genetic assays (most often fluorescence in situ hybridization and reverse transcription-polymerase chain reaction). This review discusses selected novel molecular and immunohistochemical assays with diagnostic applicability in mesenchymal neoplasms, with emphasis on diagnosis, refinement of tumor classification, and treatment stratification.


Assuntos
Neoplasias de Tecidos Moles/diagnóstico , DNA Helicases/análise , Fusão Gênica , Humanos , Imuno-Histoquímica , Proteínas Nucleares/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteína EWS de Ligação a RNA/genética , Receptor trkA/genética , Proteínas Repressoras/genética , Proteína SMARCB1/análise , Neoplasias de Tecidos Moles/química , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/patologia , Fatores de Transcrição/análise
2.
Anal Chim Acta ; 1086: 103-109, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31561784

RESUMO

Ratiometric signal transducing strategies can improve the precision of immunoassay, which possess the unique merits of spatial-resolved signal readout and self-correcting towards possible false positive results. In this work, a new ratiometric electrochemical immunosensor has been developed for reliable detection of Nuclear matrix protein 22 (NMP22). Bioinspired synthetic melanin nanospheres (SMNPs) were elaborately chosen for the following considerations: 1) SMNPs can supply good biocompatible biorecognition interface for antibody anchoring; 2) SMNPs can chelate a large amount of lead ions (Pb2+) and copper ions (Cu2+) to fabricate two spatial-resolved electrochemical signals with different response manners towards NMP22. SMNPs chelated with Pb2+ were used for the immobilization of captured primary anti-NMP22. And SMNPs chelated Cu2+ were employed to prepare signal labels after anchored with anti-NMP22 antibody. After sandwich-type immunoreaction, with the increasing concentration of NMP22, the stripping peak current of Pb2+ decreases while the stripping peak current of Cu2+ increases. Thus, ratiometric electrochemical signals could be provided for NMP22 detection. The immunosensor presented a linear concentration range from 0.013 U mL-1 to 6.7 U mL-1 with a limit of detection of 0.005 U mL-1 towards NMP22. Meanwhile, satisfactory reproducibility, stability and selectivity of the immunosensor were demonstrated.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Imunoensaio , Proteínas Nucleares/análise , Humanos , Melaninas/síntese química , Melaninas/química , Nanosferas/química
3.
Biomed Khim ; 65(4): 294-305, 2019 Jun.
Artigo em Russo | MEDLINE | ID: mdl-31436170

RESUMO

HL-60 promyelocytic cells are a widely used as a model for studying induced granulocytic differentiation. Investigation of proteins of the nuclear fraction, particularly transcription factors, is necessary for a better understanding of molecular mechanisms of cell maturation. Mass spectrometry is a powerful tool for analyzing a proteome due to its high sensitivity, specificity and performance. In this paper, using the selected reaction monitoring (SRM) method, we have assessed the levels of RBPJ, STAT1, CEBPB, CASP3, VAV1, PRKDC, PARP1 and UBC9 nuclear proteins isolated using hypertonic buffer, detergents (sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and fissionable detergent ProteaseMAX™) and using centrifugation in a sucrose density gradient. The minimum and maximum protein content was 1.13±0.28 and 14.34±1.63 fmol/mkg of total protein for the transcription factor RBPJ and ubiquitin-protein ligase type I UBC9, respectively. According to the results of shotgun mass spectrometric analysis of nuclear fractions, 2356 proteins were identified, of which 106 proteins were annotated as transcription factors. 37 transcription factors were uniquely identified in the fraction obtained by centrifugation in a sucrose density gradient, while only 9 and 8 transcription factors were uniquely identified in the nuclear fractions obtained using hypertonic buffer and detergents, respectively. The transcription factors identified in the HL-60 cell line represent regulatory molecules; their directed profiling under the influence of differentiation inducers, will shed light on the mechanism of granulocyte maturation.


Assuntos
Proteínas Nucleares/análise , Proteoma/análise , Proteômica , Fatores de Transcrição/análise , Células HL-60 , Humanos , Espectrometria de Massas
4.
Mol Med Rep ; 20(2): 1747-1753, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257492

RESUMO

Arginine/serine­rich coiled coil 1 (RSRC1) is a gene which plays a significant role in the constitutive and alternative splicing of mRNA and transcriptional regulation. It has been implicated in various neurological disorders, as well as in cancer. However, its role in gastric cancer (GC) remains unknown. Thus, the present study aimed to investigate the role of RSRC1 in GC. RSRC1 expression in GC tissues was determined by RT­qPCR and immunohistochemical staining. The effects of RSRC1 on cell proliferation and migration were detected using a Cell Counting Kit­8 assay, 5­ethynyl­2'­deoxyuridine (EdU) incorporation assay and a Transwell migration assay. Western blot analysis and RT­qPCR were used to explore the molecular mechanisms of of action of RSRC1 in GC. The results indicated that RSRC1 expression was downregulated in GC tissues compared to paired normal tissues and the reduced expression of RSRC1 was shown to contribute to a poor prognosis of patients with GC. RSRC1 knockdown promoted the proliferation and migration of GC cells. In addition, the knockdown of RSRC1 decreased the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a potent tumor suppressor gene controlling cellular growth and viability. On the whole, the findings of the present study indicate that RSRC1 functions as a tumor suppressor gene in GC and that it may exert its effects by regulating PTEN expression.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Proteínas Nucleares/análise , PTEN Fosfo-Hidrolase/análise , Neoplasias Gástricas/patologia
5.
Anal Bioanal Chem ; 411(23): 6021-6029, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31289898

RESUMO

Cytobiological methods for cell nucleus-related studies start with the extraction processes of intranuclear components with many cell lysis buffers, following with the structural characterizations and quantitative analysis of the extracted components. In this study, we tried to evaluate the availability and reliability of these extraction-based analytical methods from their spectral features. We implemented an in situ surface-enhanced Raman scattering spectroscopy (SERS) strategy with the help of the nucleus-targeting nanoprobes to investigate the molecular information of nucleus, in comparison with these ex situ methods. This study provides valuable references for choosing an appropriate detection method according to different detection purposes, and also points out the risks of many developing cell-related analytical methods that combine the traditional cytobiological techniques from exogenous interferences during sample preprocesses. Graphical abstract.


Assuntos
Núcleo Celular/química , DNA/análise , Nanopartículas Metálicas/química , Proteínas Nucleares/análise , Análise Espectral Raman/métodos , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Ouro/química , Células Hep G2 , Humanos , Nanopartículas Metálicas/ultraestrutura , Peptídeos/química , Polietilenoglicóis/química
6.
Virchows Arch ; 475(4): 407-414, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31201505

RESUMO

Nuclear membrane proteins reportedly play important roles in maintaining nuclear structures and coordinating cell activities. Studying profiles of nuclear membrane proteins may help us evaluate the biological and/or clinical nature of malignant tumors. Using immunohistochemistry with antibodies for emerin, lamin A/C, lamin B, and LAP2, we examined 105 lung cancer tissues from 33 small cell lung carcinomas (SCLCs) and 72 non-SCLCs (34 adenocarcinomas, 30 squamous cell carcinomas, and 8 large cell carcinomas). Emerin had negative or local/weak positivity in 79% of SCLCs and 1% of non-SCLCs, and lamin A/C had similar positivity in 91% of SCLCs and 3% of non-SCLCs. LAP2's expression was similar between SCLCs and non-SCLCs. RT-PCR analyses for these four nuclear membrane proteins over 7 cell lines showed that mRNA of emerin and lamin A/C were distinctly downregulated in the SCLC cell lines, supporting the immunohistochemical results. In conclusion, we suggest that downregulation of the nuclear membrane proteins emerin and lamin A/C is characteristic of SCLC cells, and this constitutional abnormality of the nuclear membrane may be related to the biological and/or clinical nature of SCLC. In addition, knowing the nuclear protein profile in SCLC cells may contribute to our understanding of nuclear fragility known as the crush artifact in pulmonary biopsy specimens.


Assuntos
Lamina Tipo A/biossíntese , Neoplasias Pulmonares/patologia , Proteínas de Membrana/biossíntese , Proteínas Nucleares/biossíntese , Carcinoma de Pequenas Células do Pulmão/patologia , Biomarcadores Tumorais/análise , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/biossíntese , Humanos , Lamina Tipo A/análise , Lamina Tipo B/análise , Lamina Tipo B/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/análise , Proteínas Nucleares/análise , Carcinoma de Pequenas Células do Pulmão/metabolismo
7.
Inflammation ; 42(4): 1389-1400, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31041569

RESUMO

Chronic nasal sinusitis with nasal polyps (CRSwNP) is a reversible nasal mucosal remodeling disease caused by persistent inflammation and structural changes in chronic nasal mucosa. Although there have been many studies on the inflammation of the nasal mucosa epithelium, the mechanism remains unclear. Our study found that H3K4me3 histone demethylase KDM2B (also known as JHDM1B) and transcriptional regulator Brg1 (also called SNF2-ß or Smarca4) were significantly decreased in nasal mucosa of CRSwNP patients, and they were positively correlated. Brg1 and KDM2B co-localize in the epithelial cells of nasal mucosa. We used poly(I:C)-treated nasal mucosal epithelial cells (HNECs) to find that the expression of KDM2B and Brg1 was also decreased, and the main expression position transferred from the nucleus to the nuclear membrane. We used small interfering RNA to knock down the expression of KDM2B and Brg1 in nasal epithelial cells. It was interesting to find that the decreased expression of KDM2B and Brg1 produced similar effects to that of poly(I:C)-treated cells, which could promote inflammatory response of nasal mucosal epithelial cells. And Brg1 appears to play a role in KDM2B regulating gene promoters of IL-6 and TNF-α inflammatory. This study shows that KDM2B and Brg1 may have an inhibitory effect on the development of CRSwNP nasal mucosal epithelial inflammation. This study will provide a new perspective for gene targeting therapy of CRSwNPs.


Assuntos
DNA Helicases/fisiologia , Proteínas F-Box/fisiologia , Inflamação/patologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Mucosa Nasal/patologia , Pólipos Nasais/etiologia , Proteínas Nucleares/fisiologia , Sinusite/complicações , Fatores de Transcrição/fisiologia , Doença Crônica , DNA Helicases/análise , Células Epiteliais/química , Células Epiteliais/patologia , Proteínas F-Box/análise , Regulação da Expressão Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/análise , Mucosa Nasal/química , Proteínas Nucleares/análise , Fatores de Transcrição/análise
8.
Curr HIV Res ; 17(1): 42-52, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31057110

RESUMO

BACKGROUND: Due to the persistence of latent HIV-infected cellular reservoirs, HIV virus can not be eradicated completely. OBJECTIVE: To identify proteins related to HIV latency, we performed a subcellular proteomic study in HIV latent cell lines. METHODS: An established HIV-1 latent cell model (J-Lat Tat-GFP Clone A7 cells, A7 cells) and its parental cell line (Jurkat cells) were used. The plasma membrane (PM) fraction from cultured cells was enriched through aqueous two-phase partition. PM proteins were extracted and then separated using two-dimensional electrophoresis (2DE). Differentially expressed proteins were identified by mass spectrometry, and verified by western blotting. RESULTS: Thirteen non-redundant proteins were identified to be differentially expressed in the A7 PM fraction compared to those in the Jurkat PM. Eight had a PM location through Gene Ontology (GO) analysis. A differential protein network of CAPG-ACTR3-CD3D was detected to have interactions with HIV Vpr, Tat, gp160, etc. through STRING software analysis. One of the differential proteins (Macrophage-capping protein (CAPG)) was verified by western blotting to be down- regulated in two cell lines and HIV resting CD4+ T cells negatively selected from patients. CONCLUSION: We identified 13 proteins in A7 compared to Jurkat cells. CAPG may be a potential biomarker related to HIV latency.


Assuntos
Membrana Celular/química , Infecções por HIV/patologia , HIV-1/fisiologia , Proteínas dos Microfilamentos/análise , Proteínas Nucleares/análise , Proteoma/análise , Linfócitos T/química , Latência Viral , Humanos , Células Jurkat
9.
Biochem Biophys Res Commun ; 511(3): 631-636, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30826064

RESUMO

Reduced expression of the Y14 gene is a cause of Thrombocytopenia-absent radius (TAR) syndrome. This gene contains a conserved RNA recognition motif (RRM) in the central region and nuclear localization/export sequences (NLS/NES) in the N-terminal. Y14 and Magoh proteins form tight heterodimers and are the core of exon junction complexes (EJCs), which mediate various processes of mRNA metabolism after transcription. In this report, we found that protein expression levels of exogenously expressed Magoh L136R and Y14 L118R (leucine-to-arginine substitution at amino acid residue 136 and 118 respectively, that results in the formation of the complex being lost) are lower than their wild-types. This reduction is likely caused by protein levels, as no difference in mRNA levels was detected. Meanwhile, a cycloheximide chase assay determined that the degradation rates of Magoh L136R and Y14 L118R were faster than their wild-types. Both Y14 L118R and Magoh L136R lost the ability to form heterodimers with corresponding wild-type proteins. However, Y14 L118R is able to still localize in the nucleus which causes the stability of Y14 L118R to be higher than Magoh L136R. These results reveal that the stability of Magoh and Y14 is not only dependent on the heterodimer structure, but also dependent on nuclear localization.


Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Núcleo Celular/genética , /metabolismo , Humanos , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Mutação Puntual , Multimerização Proteica , Estabilidade Proteica , Proteólise , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/genética , Rádio (Anatomia)/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismo , Deformidades Congênitas das Extremidades Superiores/genética , Deformidades Congênitas das Extremidades Superiores/metabolismo
10.
Mol Med Rep ; 19(3): 2125-2136, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30747208

RESUMO

AT­rich interaction domain 1A (ARID1A) and phosphatidylinositol­4,5­bisphosphate 3­kinase catalytic subunit α (PIK3CA) serve important roles in the formation and development of numerous malignancies including gastric cancer. Accumulating evidence has demonstrated that Epstein­Barr virus (EBV) is a pathogenic virus associated with gastric cancer. The present study aimed to investigate the association between EBV infection, and the expression levels of ARID1A and PIK3CA in gastric cancer. EBER in situ hybridization was performed to detect EBV infection. Immunohistochemistry was used to assess the expression levels of ARID1A and PIK3CA in gastric cancer and adjacent normal tissues. A total of 58 gastric cancer and 10 adjacent normal tissues were tested for genetic mutations via single nucleotide polymorphism genotyping assays. Fluorescent polymerase chain reaction was used to detect EBV infection; 9.3% (28/300) of gastric cancer samples were positive for EBV, whereas, all adjacent normal tissues were negative. ARID1A and PIK3CA were negatively correlated in gastric cancer (r=­0.167). The expression levels of ARID1A and PIK3CA in gastric cancer were significantly associated with the depth of invasion of gastric cancer. A total of 62.1% (36/58) of tumor samples exhibited mutations in ARID1A, whereas, 13.8% (8/58) presented mutations in PIK3CA. Notably, EBV­associated gastric cancer (EBVaGC) samples with PIK3CA mutations additionally exhibited ARID1A mutations. Although in the present study it was identified that ARID1A and PIK3CA were negatively correlated in EBVaGC, further studies are required to investigate the association among ARID1A, PIK3CA and EBV in gastric cancer.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Infecções por Vírus Epstein-Barr/complicações , Herpesvirus Humano 4/isolamento & purificação , Proteínas Nucleares/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/virologia , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Classe I de Fosfatidilinositol 3-Quinases/análise , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/análise , Polimorfismo de Nucleotídeo Único , Neoplasias Gástricas/patologia , Fatores de Transcrição/análise , Adulto Jovem
11.
Virchows Arch ; 475(1): 85-94, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30739164

RESUMO

Several subtypes of high-grade endometrial carcinomas (ECs) contain an undifferentiated component of non-epithelial morphology, including undifferentiated and dedifferentiated carcinomas and carcinosarcomas (CSs). The mechanism by which an EC undergoes dedifferentiation has been the subject of much debate. The epithelial-mesenchymal transition (EMT) is one of the mechanisms implicated in the transdifferentiation of high-grade carcinomas. To improve our understanding of the role of EMT in these tumors, we studied a series of 89 carcinomas including 14 undifferentiated/dedifferentiated endometrial carcinomas (UECs/DECs), 49 CSs (21 endometrial, 29 tubo-ovarian and peritoneal), 17 endometrioid carcinomas (grade 1-3), and 9 high-grade serous carcinomas of the uterus, using a panel of antibodies targeting known epithelial markers (Pan-Keratin AE1/AE3 and E-cadherin), mesenchymal markers (N-cadherin), EMT transcription factors (TFs) (ZEB1, ZEB2, TWIST1), PAX8, estrogen receptors (ER), progesterone receptors (PR), and the p53 protein. At least one of the three EMT markers (more frequently ZEB1) was positive in the sarcomatous component of 98% (n = 48/49) of CSs and 98% (n = 13/14) of the undifferentiated component of UEC/DEC. In addition, 86% of sarcomatous areas of CSs and 79% of the undifferentiated component of UEC/DEC expressed all three EMT-TFs. The expression of these markers was associated with the loss of or reduction in epithelial markers (Pan-keratin, E-cadherin), PAX8, and hormone receptors. In contrast, none of the endometrioid and serous endometrial carcinomas expressed ZEB1, while 6% and 36% of endometrioid and 11% and 25% of serous carcinomas focally expressed ZEB2 and TWIST1, respectively. Although morphologically different, EMT appears to be implicated in the dedifferentiation in both CSs and UEC/DEC. Indeed, we speculate that the occurrence of EMT in a well differentiated endometrioid carcinoma may consecutively lead to a dedifferentiated and undifferentiated carcinoma, while in a type II carcinoma, it may result in a CS.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma/química , Transição Epitelial-Mesenquimal , Neoplasias Uterinas/química , Carcinoma/classificação , Carcinoma/patologia , Carcinoma Endometrioide/química , Carcinoma Endometrioide/patologia , Carcinossarcoma/química , Carcinossarcoma/patologia , Desdiferenciação Celular , Neoplasias do Endométrio/química , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Gradação de Tumores , Proteínas Nucleares/análise , Estudos Retrospectivos , Proteína 1 Relacionada a Twist/análise , Neoplasias Uterinas/classificação , Neoplasias Uterinas/patologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/análise , Homeobox 1 de Ligação a E-box em Dedo de Zinco/análise
12.
World J Gastroenterol ; 25(1): 138-150, 2019 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-30643364

RESUMO

AIM: To evaluate the clinical properties of three subpopulations of circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) patients. METHODS: We identified CTCs for expression of the epithelial cell marker cytokeratin or epithelial cell adhesion molecule (EpCAM) (E-CTC), the mesenchymal cell markers vimentin and twist (M-CTC), or both (E/M-CTC) using the CanPatrol system. Between July 2014 and July 2016, 107 patients with PDAC were enrolled for CTC evaluation. CTC enumeration and classification were correlated with patient clinicopathological features and outcomes. RESULTS: CTCs were detected in 78.5% of PDAC patients. The number of total CTCs ranged from 0 to 26 across all 107 patients, with a median value of six. CTC status correlated with lymph node metastasis, TNM stage, distant metastasis, blood lymphocyte counts, and neutrophil-to-lymphocyte ratio (NLR). Kaplan-Meier survival analysis showed that patients with ≥ 6 total CTCs had significantly decreased overall survival and progression-free survival compared with patients with < 6 total CTCs. The presence of M-CTCs was positively correlated with TNM stage (P < 0.01) and distant metastasis (P < 0.01). Additionally, lymphocyte counts and NLR in patients without CTCs were significantly different from those in patients testing positive for each CTC subpopulation (P < 0.01). CONCLUSION: Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulations and provides useful evidence for determining a suitable clinical approach.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma Ductal Pancreático/patologia , Transição Epitelial-Mesenquimal , Células Neoplásicas Circulantes/patologia , Neoplasias Pancreáticas/patologia , Idoso , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/mortalidade , Molécula de Adesão da Célula Epitelial/análise , Feminino , Humanos , Estimativa de Kaplan-Meier , Biópsia Líquida/métodos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/análise , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/mortalidade , Prognóstico , Proteína 1 Relacionada a Twist/análise , Vimentina/análise
13.
Nucleic Acids Res ; 47(6): e31, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30657937

RESUMO

High-throughput (HT) in vitro methods for measuring protein-DNA binding have become invaluable for characterizing transcription factor (TF) complexes and modeling gene regulation. However, current methods do not utilize endogenous proteins and, therefore, do not quantify the impact of cell-specific post-translational modifications (PTMs) and cooperative cofactors. We introduce the HT nextPBM (nuclear extract protein-binding microarray) approach to study DNA binding of native cellular TFs that accounts for PTMs and cell-specific cofactors. We integrate immune-depletion and phosphatase treatment steps into our nextPBM pipeline to characterize the impact of cofactors and phosphorylation on TF binding. We analyze binding of PU.1/SPI1 and IRF8 from human monocytes, delineate DNA-sequence determinants for their cooperativity, and show how PU.1 affinity correlates with enhancer status and the presence of cooperative and collaborative cofactors. We describe how nextPBMs, and our accompanying computational framework, can be used to discover cell-specific cofactors, screen for synthetic cooperative DNA elements, and characterize TF cooperativity.


Assuntos
Núcleo Celular/química , Redes Reguladoras de Genes , Análise Serial de Proteínas/métodos , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Extratos Celulares/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Células THP-1
14.
J Cancer Res Clin Oncol ; 145(3): 661-673, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30643969

RESUMO

PURPOSE: We aimed to analyze the expression of ZWINT, NUSAP1, DLGAP5, and PRC1 in tumor tissues and adjacent tissues with public data. METHODS: The expression patterns of four genes were detected in cancer tissues and adjacent tissues by qRT-PCR. The overall survival analysis was used to explore these genes in lung adenocarcinoma and squamous cell carcinoma patients. Knockdown assays were used to select the most suitable gene among these four genes. Cell function assays with the knockdown gene were conducted in A549 and NCL H226 cells. The role of the knockdown gene in lung cancer was dissected in a mice tumor model. Transcriptome sequencing analyses with the knockdown gene were analyzed. RESULTS: Overexpression of these genes was significantly detected in cancer tissues (P < 0.01). Overall survival revealed that high expression of these genes is closely related with poor prognosis of lung adenocarcinoma patients (P < 0.05). Knockdown of ZWINT reduced proliferation in NCI H226 and A549 cells (P < 0.05). Knockdown also inhibited cell migration, invasion, apoptosis, and colony formation (P < 0.05). ZWINT knockdown reduced tumor volume (P < 0.05). Transcriptome sequencing of ZWINT knockdown-treated A549 and NCI H226 cells indicated that 100 and 426 differentially expressed genes were obtained, respectively. Gene ontology analysis suggested that binding, biological regulation, and multicellular organismal processes were the most enriched. KEGG analysis revealed that TNF, P53, and PI3K signal networks would be the most potential ZWINT-related pathways and were identified by Western blot analysis. CONCLUSIONS: ZWINT may be a novel target for lung cancer therapy.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/patologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Neoplasias Pulmonares/patologia , Proteínas Nucleares/biossíntese , Animais , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares/análise
15.
Methods Enzymol ; 616: 365-383, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30691651

RESUMO

Genome organization and subnuclear protein localization are essential for normal cellular function and have been implicated in the control of gene expression, DNA replication, and genomic stability. The coupling of chromatin conformation capture (3C), chromatin immunoprecipitation and sequencing, and related techniques have continuously improved our understanding of genome architecture. To profile site-specifically DNA-associated proteins in a high-throughput and unbiased manner, the RNA-programmable CRISPR-Cas9 platform has recently been combined with an enzymatic labeling system to allow proteomic landscapes at repetitive and nonrepetitive loci to be defined with unprecedented ease and resolution. In this chapter, we describe the dCas9-APEX2 experimental approach for specifically targeting a DNA sequence, enzymatically labeling local proteins with biotin, and quantitatively analyzing the labeled proteome. We also discuss the optimization and extension of this pipeline to facilitate its use in understanding nuclear and chromosome biology.


Assuntos
Ascorbato Peroxidases/genética , Proteína 9 Associada à CRISPR/genética , Proteínas Nucleares/análise , Proteômica/métodos , Soja/enzimologia , Sistemas CRISPR-Cas , Clonagem Molecular/métodos , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Espectrometria de Massas/métodos , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Soja/genética
16.
Cancer Immunol Immunother ; 68(3): 443-454, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30604042

RESUMO

Adenocarcinoma of the ampulla of Vater (AOV) is classified into intestinal type (IT) and pancreatobiliary type (PB); however, the immunological properties of these subtypes remain to be characterized. Here, we evaluated the clinical implications of PD-L1 expression and CD8+ T lymphocyte density in adenocarcinomas of the AOV and their potential association with Yes-associated protein (YAP). We analyzed 123 adenocarcinoma-of-the-AOV patients who underwent surgical resection, and tumors were classified into IT or PB type. Tumor or inflammatory cell PD-L1 expression, CD8+ T lymphocyte density in the cancer cell nest (intratumoral) or in the adjacent stroma, and YAP localization and intensity were analyzed using immunohistochemical staining. PB-type tumors showed higher tumoral PD-L1 expression than IT-type tumors, and tumoral PD-L1 expression was associated with a shorter disease-free survival (DFS) [hazard ratio (HR), 1.77; p = 0.045] and overall survival (OS) (HR 1.99; p = 0.030). Intratumoral CD8+ T lymphocyte density was higher in IT type than in PB type and was associated with a favorable DFS (HR 0.47; p = 0.022). The nuclear staining pattern of YAP in tumor cells, compared to non-nuclear staining patterns, was more frequently associated with PB type and increased tumoral PD-L1 expression. Nuclear YAP staining was a significant prognostic factor for OS (HR 2.21; p = 0.022). These results show that the two subtypes of adenocarcinoma of the AOV exhibit significant differences in tumoral PD-L1 expression and intratumoral CD8+ T lymphocyte density, which might contribute to their distinct clinical features.


Assuntos
Adenocarcinoma/imunologia , Ampola Hepatopancreática , Neoplasias do Ducto Colédoco/imunologia , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Antígeno B7-H1/análise , Linfócitos T CD8-Positivos/imunologia , Proteínas de Ciclo Celular , Neoplasias do Ducto Colédoco/mortalidade , Neoplasias do Ducto Colédoco/patologia , Feminino , Humanos , Imuno-Histoquímica , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/análise , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Transcrição/análise
17.
Analyst ; 144(2): 649-655, 2019 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-30480684

RESUMO

In this study, a new, simple, and label-free electrochemical immunosensor was presented for the detection of nuclear matrix protein-22 (NMP-22). In order to accurately monitor very small amounts of NMP-22, it was advantageous to use highly efficient nanomaterials as signals. For this reason, we synthesized a chrysanthemum-like nanocomposite (Co-MOFs/CuAu NWs), using Co-based metal-organic frameworks (Co-MOFs) as carriers and copper gold nanowires (CuAu NWs) wrapped around their surface, which was applied for modifying a glassy carbon electrode (GCE). The Co-MOFs/CuAu NWs possessed outstanding catalytic capabilities, which served as signal materials and simultaneously carried the anti-NMP-22 antibody (Ab). When different concentrations of the NMP-22 antigen (Ag) were specifically attached to the immunosensor, the current responses decreased by varying degrees. The designed biosensor used the principle to establish a linear regression equation and achieve an accurate quantification of NMP-22. After optimization, the NMP-22 sensor exhibited a good linear response over a concentration range from 0.1 pg mL-1 to 1 ng mL-1, with a lower detection limit of 33 fg mL-1 (based on S/N = 3). The proposed biosensor demonstrated the advantages of ultra-sensitivity, high specificity and acceptable reproducibility, suggesting that the proposed strategy has the potential for the quantification of NMP-22 in human urine samples. Moreover, the novel nanocomposite Co-MOFs/CuAu NWs are promising materials for electrochemical sensors to detect other biomolecules.


Assuntos
Técnicas Biossensoriais/métodos , Imunoensaio/métodos , Estruturas Metalorgânicas/química , Metais Pesados/química , Nanocompostos/química , Proteínas Nucleares/análise , Anticorpos Imobilizados/química , Cobalto/química , Cobre/química , Eletroquímica , Eletrodos , Ouro/química , Humanos , Limite de Detecção , Proteínas Nucleares/urina
18.
Cell Mol Life Sci ; 76(1): 129-145, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30151693

RESUMO

As an analgesic and antipyretic drug, acetaminophen (APAP) is commonly used and known to be safe at therapeutic doses. In many countries, the overuse of APAP provokes acute liver injury and even liver failure. APAP-induced liver injury (AILI) is the most used experimental model of drug-induced liver injury (DILI). Here, we have demonstrated elevated levels of growth arrest and DNA damage-inducible 45α (GADD45α) in the livers of patients with DILI/AILI, in APAP-injured mouse livers and in APAP-treated hepatocytes. GADD45α exhibited a protective effect against APAP-induced liver injury and alleviated the accumulation of small lipid droplets in vitro and in vivo. We found that GADD45α promoted the activation of AMP-activated protein kinase α and induced fatty acid beta-oxidation, tricarboxylic acid cycle (TCA) and glycogenolysis-related gene expression after APAP exposure. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that GADD45α increased the levels of TCA cycle metabolites. Co-immunoprecipitation analysis showed that Ppp2cb, a catalytic subunit of protein phosphatase 2A, could interact directly with GADD45α. Our results indicate that hepatocyte GADD45α might represent a therapeutic target to prevent and rescue liver injury caused by APAP.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetaminofen/efeitos adversos , Antipiréticos/efeitos adversos , Proteínas de Ciclo Celular/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fígado/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Proteínas Quinases Ativadas por AMP/análise , Analgésicos não Entorpecentes/efeitos adversos , Animais , Proteínas de Ciclo Celular/análise , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/análise , Transdução de Sinais/efeitos dos fármacos
19.
J Biol Chem ; 294(2): 576-592, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30409912

RESUMO

Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore-microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore-microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80-microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1-80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80-microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore-microtubule attachment in the maintenance of genomic stability during mitosis.


Assuntos
Cinetocoros/metabolismo , Lisina Acetiltransferase 5/metabolismo , Microtúbulos/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Acetilação , Segregação de Cromossomos , Proteínas do Citoesqueleto , Células HEK293 , Células HeLa , Humanos , Lisina Acetiltransferase 5/análise , Modelos Moleculares , Proteínas Nucleares/análise , Mapas de Interação de Proteínas , Sirtuína 1/análise , Sirtuína 1/metabolismo
20.
Curr Opin Chem Biol ; 48: 1-7, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30170243

RESUMO

Protein functions are tightly regulated by their subcellular localization and dynamic alteration. Chemical proteomics offers convenience and efficiency for profiling protein features in a native context. In this review, we summarize the recent progress of subcellular-compartment-focused chemical proteomics which do not rely on organelle fractionation. Organelle-specific activity-based protein profiling (ABPP) and engineered ascorbate peroxidase (APEX) have been developed for proteome analysis within organelles and even sub-organelles. In parallel, our lab designed organelle-localizable reactive molecules (ORMs) to selectively tag nuclear and mitochondrial proteins. ORMs-based proteomics is applicable to primary neurons and brain slices, as well as cultured cell lines. In addition, we invented a conditional proteomics approach to elucidate zinc homeostasis by labeling and identifying proteins localized in Zn2+-rich space of live cells.


Assuntos
Proteínas Mitocondriais/análise , Proteínas Nucleares/análise , Organelas/metabolismo , Proteoma/análise , Proteômica/métodos , Animais , Humanos , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Organelas/química , Proteoma/metabolismo , Zinco/análise , Zinco/metabolismo
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