Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 17.330
Filtrar
1.
Nihon Yakurigaku Zasshi ; 154(4): 186-191, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31597897

RESUMO

Primary cilia, which protrude from the surfaces of most human cells, function as cellular antennae that receive extracellular signals. To serve as antennae, primary cilia contain unique proteins, such as G-protein-coupled receptors and ion channels. Defects in the assembly and functions of primary cilia cause hereditary disorders with a wide range of symptoms, including cystic kidney and retinal degeneration. The assembly and maintenance of cilia depend on protein trafficking mediated by the intraflagellar transport (IFT) machinery, which contains three protein complexes (IFT-A, IFT-B, and BBSome) and two motor proteins (kinesin-2 and dynein-2 complex) and is composed of more than 40 subunits in total. We recently revealed the interaction between the kinesin-2 and IFT-B complexes and overall architecture of the dynein-2 complex by taking advantage of the visible immunoprecipitation (VIP) assay. In addition, we clarified the roles of dynein-2 subunits using gene knockout cell lines established using the CRISPR/Cas9 system. This review focuses on recent advances in the architectures and functions of two motor proteins underlying the IFT machinery.


Assuntos
Cílios/fisiologia , Dineínas/fisiologia , Flagelos/fisiologia , Cinesina/fisiologia , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Imunoprecipitação , Transporte Proteico
2.
Nat Protoc ; 14(10): 2749-2780, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31471598

RESUMO

Circulating cell-free DNA (cfDNA) comprises small DNA fragments derived from normal and tumor tissue that are released into the bloodstream. Recently, methylation profiling of cfDNA as a liquid biopsy tool has been gaining prominence due to the presence of tissue-specific markers in cfDNA. We have previously reported cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) as a sensitive, low-input, cost-efficient and bisulfite-free approach to profiling DNA methylomes of plasma cfDNA. cfMeDIP-seq is an extension of a previously published MeDIP-seq protocol and is adapted to allow for methylome profiling of samples with low input (ranging from 1 to 10 ng) of DNA, which is enabled by the addition of 'filler DNA' before immunoprecipitation. This protocol is not limited to plasma cfDNA; it can also be applied to other samples that are naturally sheared and at low availability (e.g., urinary cfDNA and cerebrospinal fluid cfDNA), and is potentially applicable to other applications beyond cancer detection, including prenatal diagnostics, cardiology and monitoring of immune response. The protocol presented here should enable any standard molecular laboratory to generate cfMeDIP-seq libraries from plasma cfDNA in ~3-4 d.


Assuntos
Ácidos Nucleicos Livres/análise , Ácidos Nucleicos Livres/metabolismo , Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ácidos Nucleicos Livres/isolamento & purificação , Biologia Computacional/métodos , Biblioteca Gênica , Humanos , Imunoprecipitação , Fluxo de Trabalho
3.
BMC Bioinformatics ; 20(1): 414, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387525

RESUMO

BACKGROUND: R-loops are three-stranded nucleic acid structures that usually form during transcription and that may lead to gene regulation or genome instability. DRIP (DNA:RNA Immunoprecipitation)-seq techniques are widely used to map R-loops genome-wide providing insights into R-loop biology. However, annotation of DRIP-seq peaks to genes can be a tricky step, due to the lack of strand information when using the common basic DRIP technique. RESULTS: Here, we introduce DRIP-seq Optimized Peak Annotator (DROPA), a new tool for gene annotation of R-loop peaks based on gene expression information. DROPA allows a full customization of annotation options, ranging from the choice of reference datasets to gene feature definitions. DROPA allows to assign R-loop peaks to the DNA template strand in gene body with a false positive rate of less than 7%. A comparison of DROPA performance with three widely used annotation tools show that it identifies less false positive annotations than the others. CONCLUSIONS: DROPA is a fully customizable peak-annotation tool optimized for co-transcriptional DRIP-seq peaks, which allows a finest gene annotation based on gene expression information. Its output can easily be integrated into pipelines to perform downstream analyses, while useful and informative summary plots and statistical enrichment tests can be produced.


Assuntos
DNA/metabolismo , Imunoprecipitação , Anotação de Sequência Molecular , RNA/metabolismo , Software , Sequência de Bases , DNA/genética , Regulação da Expressão Gênica , RNA/genética
4.
Cell Physiol Biochem ; 53(3): 465-479, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31464387

RESUMO

BACKGROUND/AIMS: Cyclophilin D (CypD) mediates the mitochondrial permeability transition pore (mPTP) opening that contributes to mitochondrial dysfunction. CypD is regulated by its acetylation/deacetylation state that depends on Sirtuin-3 (SIRT3) mitochondrial deacetylase. Since obesity and metabolic syndrome decrease SIRT3 activity and expression, we tested the hypothesis that CypD hyperacetylation promotes mitochondrial dysfunction under this pathophysiological state, which is associated with ventricular dysfunction and heart failure. METHODS: Myocardial tissue samples from patients with left ventricular heart failure, with either obesity or normal weight, were processed for the expression of SIRT3 and acetylation profile by Western Blot (WB). In addition, a rat model of obesity and metabolic syndrome induced by 30% (w/v) of sucrose was conducted. The WB analysis was used to determine the levels of mitochondrial expression of SIRT3, Adenine Nucleotide Translocator (ANT), CypD and the acetylation profile, as well as immunoprecipitation to establish the acetylation levels of CypD. Mitochondrial function was assessed by oxygen consumption analysis and maximum Ca2+ retention capacity. Oxidative stress was assessed by aconitase activity, protein carbonyl and thiol groups content. RESULTS: SIRT3 expression in the biopsies of the failing human hearts showed a 46% decrease in the expression levels of obese patients in comparison to the non-obese patients (p=0.0219). Remarkably, body mass index was associated with protein acetylation (0.627; p = 0.035), suggesting that the acetylation profiles of the failing hearts of obese patients are partly mediated by a reduction in SIRT3, which is also associated with higher BNP levels, indicating a more severe ventricular dysfunction (-0.636; p = 0.043). Accordingly, obese rats demonstrated a SIRT3 mitochondrial expression decrease of 22% concomitantly with a hyperacetylated mitochondrial profile, including CypD. Cardiac mitochondria from obese animals were 2.5-fold more prone to mPTP opening than the controls. CONCLUSION: Our results indicate that obesity reduces SIRT3 expression and that CypD hyperacetylation increases mPTP opening, suggesting that the activation of SIRT3 might be a potential target to decrease ventricular dysfunction and slow the progression of heart failure.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Obesidade/metabolismo , Sirtuína 3/metabolismo , Acetilação , Adulto , Idoso , Animais , Índice de Massa Corporal , Cálcio/metabolismo , Ciclofilinas/metabolismo , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Imunoprecipitação , Técnicas In Vitro , Masculino , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Translocases Mitocondriais de ADP e ATP/metabolismo , Consumo de Oxigênio/fisiologia , Ratos , Ratos Wistar
5.
Nat Commun ; 10(1): 2394, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160584

RESUMO

To understand the molecular processes that link Aß amyloidosis, tauopathy and neurodegeneration, we screened for tau-interacting proteins by immunoprecipitation/LC-MS. We identified the carboxy-terminal PDZ ligand of nNOS (CAPON) as a novel tau-binding protein. CAPON is an adaptor protein of neuronal nitric oxide synthase (nNOS), and activated by the N-methyl-D-aspartate receptor. We observed accumulation of CAPON in the hippocampal pyramidal cell layer in the AppNL-G-F -knock-in (KI) brain. To investigate the effect of CAPON accumulation on Alzheimer's disease (AD) pathogenesis, CAPON was overexpressed in the brain of AppNL-G-F mice crossbred with MAPT (human tau)-KI mice. This produced significant hippocampal atrophy and caspase3-dependent neuronal cell death in the CAPON-expressing hippocampus, suggesting that CAPON accumulation increases neurodegeneration. CAPON expression also induced significantly higher levels of phosphorylated, oligomerized and insoluble tau. In contrast, CAPON deficiency ameliorated the AD-related pathological phenotypes in tauopathy model. These findings suggest that CAPON could be a druggable AD target.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/metabolismo , Hipocampo/metabolismo , Agregação Patológica de Proteínas/metabolismo , Células Piramidais/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Atrofia , Caspase 3/metabolismo , Morte Celular , Cromatografia Líquida , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Hipocampo/patologia , Humanos , Imunoprecipitação , Espectrometria de Massas , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Agregação Patológica de Proteínas/patologia , Células Piramidais/patologia , Tauopatias , Proteínas tau/metabolismo
6.
Mol Biol (Mosk) ; 53(3): 393-401, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184604

RESUMO

The efficiency at which specific transcription factors interact with DNA may vary in the presence of single nucleotide polymorphisms (SNPs), and the variation provides an important mechanism that regulates expression of human genes and contributes to the individual susceptibility to various diseases. Ample genetic and epigenetic data make it possible to predict both functional polymorphic variants and the transcription factors whose binding they affect. However, predictions of the kind require experimental verification. An original method developed for the purpose includes immunoprecipitation of DNA-protein complexes, followed by quantification of the bound DNA by real-time PCR. The method does not require chemical modification of the DNA probes and yield reproducible results with total nuclear extracts from cultured human cells.


Assuntos
Alelos , Imunoprecipitação , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Humanos , Ligação Proteica
7.
Exp Parasitol ; 203: 8-18, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31150653

RESUMO

Toxoplasma gondii is an important human and veterinary pathogen and the causative agent of toxoplasmosis, a potentially severe disease especially in immunocompromised or congenitally infected humans. Current therapeutic compounds are not well-tolerated, present increasing resistance, limited efficacy and require long periods of treatment. On this context, searching for new therapeutic targets is crucial to drug discovery. In this sense, recent works suggest that N-myristoyltransferase (NMT), the enzyme responsible for protein myristoylation that is essential in some parasites, could be the target of new anti-parasitic compounds. However, up to date there is no information on NMT and the extent of this modification in T. gondii. In this work, we decided to explore T. gondii genome in search of elements related with the N-myristoylation process. By a bioinformatics approach it was possible to identify a putative T. gondii NMT (TgNMT). This enzyme that is homologous to other parasitic NMTs, presents activity in vitro, is expressed in both intra- and extracellular parasites and interacts with predicted TgNMT substrates. Additionally, NMT activity seems to be important for the lytic cycle of Toxoplasma gondii. In parallel, an in silico myristoylome predicts 157 proteins to be affected by this modification. Myristoylated proteins would be affecting several metabolic functions with some of them being critical for the life cycle of this parasite. Together, these data indicate that TgNMT could be an interesting target of intervention for the treatment of toxoplasmosis.


Assuntos
Aciltransferases/metabolismo , Toxoplasma/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/efeitos dos fármacos , Aciltransferases/genética , Sequência de Aminoácidos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Fibroblastos/parasitologia , Imunofluorescência , Prepúcio do Pênis/citologia , Prepúcio do Pênis/parasitologia , Humanos , Imunoprecipitação , Masculino , Filogenia , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Toxoplasma/classificação , Toxoplasma/enzimologia , Toxoplasma/genética
8.
Gut ; 68(10): 1858-1871, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31118247

RESUMO

BACKGROUND AND AIMS: The unique expression pattern makes oncofetal proteins ideal diagnostic biomarkers and therapeutic targets in cancer. However, few oncofetal proteins have been identified and entered clinical practice. METHODS: Fetal liver, adult liver and hepatocellular carcinoma (HCC) tissues were employed to assess the expression of hepatic leukaemia factor (HLF). The impact of HLF on HCC onset and progression was investigated both in vivo and in vitro. The association between HLF and patient prognosis was determined in patient cohorts. The correlation between HLF expression and sorafenib benefits in HCC was further evaluated in patient cohorts and patient-derived xenografts (PDXs). RESULTS: HLF is a novel oncofetal protein which is reactivated in HCC by SOX2 and OCT4. Functional studies revealed that HLF transactivates c-Jun to promote tumour initiating cell (TIC) generation and enhances TIC-like properties of hepatoma cells, thus driving HCC initiation and progression. Consistently, our clinical investigations elucidated the association between HLF and patient prognosis and unravelled the close correlation between HLF levels and c-Jun expression in patient HCCs. Importantly, HLF/c-Jun axis determines the responses of hepatoma cells to sorafenib treatment, and interference of HLF abrogated c-Jun activation and enhanced sorafenib response. Analysis of patient cohorts and PDXs further suggests that HLF/c-Jun axis might serve as a biomarker for sorafenib benefits in HCC patients. CONCLUSIONS: Our findings uncovered HLF as a novel oncofetal protein and revealed the crucial role of the HLF/c-Jun axis in HCC development and sorafenib response, rendering HLF as an optimal target for the prevention and intervention of HCC.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Carcinoma Hepatocelular/genética , Resistencia a Medicamentos Antineoplásicos , Genes jun/genética , Neoplasias Hepáticas/genética , Sorafenibe/farmacologia , Adulto , Antineoplásicos/farmacologia , Apoptose , Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Progressão da Doença , Feminino , Humanos , Imunoprecipitação , Zíper de Leucina , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Masculino , Prognóstico
9.
Plant Sci ; 284: 117-126, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084864

RESUMO

Previously, we showed that transplastomic tobacco plants expressing the LiHsp83-SAG1 fusion protein displayed a chlorotic phenotype and growth retardation, while plants expressing the SAG1 and GRA4 antigens alone did not. We conducted a comprehensive examination of the metabolic and photosynthetic parameters that could be affecting the normal growth of LiHsp83-SAG1 plants in order to understand the origin of these pleiotropic effects. These plants presented all photosynthetic pigments and parameters related to PSII efficiency significantly diminished. However, the expression of CHLI, RSSU and LHCa/b genes did not show significant differences between LiHsp83-SAG1 and control plants. Total protein, starch, and soluble sugar contents were also greatly reduced in LiHsp83-SAG1 plants. Since Hsp90 s are constitutively expressed at much higher concentrations at high temperatures, we tested if the fitness of LiHsp83-SAG1 over-expressing LiHsp83 would improve after heat treatment. LiHsp83-SAG1 plants showed an important alleviation of their phenotype and an evident recovery of the PSII function. As far as we know, this is the first report where it is demonstrated that a transplastomic line performs much better at higher temperatures. Finally, we detected that LiHsp83-SAG1 protein could be binding to key photosynthesis-related proteins at 37 °C. Our results suggest that the excess of this molecular chaperone could benefit the plant in a possible heat shock and prevent the expected denaturation of proteins. However, the LiHsp83-SAG1 protein content was weakly decreased in heat-treated plants. Therefore, we cannot rule out that the alleviation observed at 37 °C may be partially due to a reduction of the levels of the recombinant protein.


Assuntos
Antígenos de Protozoários/metabolismo , Proteínas de Choque Térmico/metabolismo , Leishmania infantum/metabolismo , Fotossíntese , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Toxoplasma/metabolismo , Clorofila/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica , Temperatura Alta , Imunoprecipitação , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/parasitologia , Tabaco
10.
MBio ; 10(2)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040239

RESUMO

The tissue cyst of Toxoplasma gondii, found in latent infection, serves a critical role in both transmission and reactivation of this organism. Within infected cells, slowly replicating parasites (bradyzoites) are surrounded by a cyst matrix, cyst wall, and cyst membrane. The cyst wall is clearly delineated by ultrastructural analysis; however, the composition and function of this layer in host-parasite interactions are not fully understood. In order to understand the composition of the cyst wall, a proteomic analysis of purified cyst wall fragments, that were enriched with Percoll gradients and subsequently immunoprecipitated with CST1 antibody, was performed. Known cyst wall proteins, such as CST1, BPK1, MCP4, MAG1, GRA2, GRA3, and GRA5, were identified in this preparation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, dense granule proteins (GRAs) not previously shown to associate with the cyst wall, as well as uncharacterized hypothetical proteins, were identified in this cyst wall preparation. Several of these hypothetical cyst wall (CST) proteins were epitope tagged, and immunofluorescence assays confirmed their localization as novel cyst matrix and cyst wall proteins. Expression of two of these newly identified cyst wall proteins was eliminated by gene knockout (CST2-KO and CST3-KO). CST2-KO parasites were highly attenuated in virulence and did not establish detectable cyst burdens. This targeted proteomic approach allowed the identification of new components of the cyst wall that probably have roles in the parasite/host interface.IMPORTANCE Toxoplasma gondii is a highly prevalent parasite worldwide that presents life-threatening risks to immunocompromised and pregnant individuals. Whereas the life stage responsible for acute infection can be treated, the life stage responsible for chronic infection is refractory to currently available therapeutics. Little is known about the protein composition of the cyst wall, an amorphous structure formed by parasites that is suspected to facilitate persistence within muscle and nervous tissue during chronic (latent) infection. By implementing a refined approach to selectively purify cyst wall fragments, we identified several known and novel cyst wall proteins from our sample preparations. We confirmed the localizations of several proteins from this data set and identified one that is involved in parasite virulence. These data will propel further studies on cyst wall structure and function, leading to therapeutic strategies that can eliminate the chronic infection stage.


Assuntos
Parede Celular/química , Proteoma/análise , Proteínas de Protozoários/análise , Esporos de Protozoários/química , Toxoplasma/química , Animais , Células Cultivadas , Centrifugação com Gradiente de Concentração , Cromatografia Líquida , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Humanos , Imunoprecipitação , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Proteômica , Proteínas de Protozoários/genética , Espectrometria de Massas em Tandem , Toxoplasma/genética , Toxoplasmose/parasitologia , Toxoplasmose/patologia , Virulência
11.
Anal Bioanal Chem ; 411(14): 3009-3019, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31076819

RESUMO

The N-glycosylation of proteins is one of the most important post-translational modifications relevant to various biological functions. The identification and quantification of N-glycoproteins in liquid chromatography-mass spectrometry (LC-MS) is challenging because of their low analytical sensitivity and selectivity. This is due to their microheterogeneity and the difficulty of synthesizing N-glycopeptides as an internal standard. Parallel reaction monitoring (PRM) is widely used in targeted LC-MS. The key advantage of LC-PRM is that it can identify N-glycopeptides using tandem mass spectrometry (MS/MS) fragmentation, even without an internal standard. We investigated the feasibility of analyzing N-glycoproteins using multiplex immunoprecipitation to improve sensitivity and selectivity. We targeted N-glycoproteins [α-fetoprotein (AFP), vitronectin (VTN), and α-1-antichymotrypsin (AACT)] that are abnormally glycosylated in hepatocellular carcinoma (HCC). Their tryptic N-glycopeptides were selected to determine the percentages of fucosylated N-glycopeptides using Y ions, which include glycopeptide fragments with amino acid sequences. Finally, we confirmed that the area under the receiver operating characteristic curve (AUC = 0.944) for the combination of AFP and VTN increased more so than for a single glycopeptide (AUC = 0.889 for AFP and 0.792 for VTN) with respect to discriminating between HCC and cirrhosis serum. This study shows that an LC-PRM method using multiplex N-glycoproteins immunoprecipitated from serum could be applied to develop and verify cancer biomarkers. Graphical abstract.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/diagnóstico , Cromatografia Líquida/métodos , Glicoproteínas/sangue , Imunoprecipitação/métodos , Neoplasias Hepáticas/diagnóstico , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Calibragem , Carcinoma Hepatocelular/sangue , Estudos de Casos e Controles , Estudos de Viabilidade , Fucose/química , Glicoproteínas/química , Glicoproteínas/normas , Glicosilação , Humanos , Limite de Detecção , Neoplasias Hepáticas/sangue , Curva ROC , Padrões de Referência , Vitronectina/sangue , alfa 1-Antiquimotripsina/sangue , alfa-Fetoproteínas/metabolismo
12.
Vet Immunol Immunopathol ; 211: 19-24, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084889

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is one of the most common diseases in the global swine industry. PRRSV infection is highly restricted to cells of the monocyte-macrophage lineage. However, the lack of antibodies to swine monocyte-macrophage lineage markers significantly hampers PRRSV research. In this study, we have developed a monoclonal antibody against the swine leukocyte antigen (SLA)-DRα chain and confirmed its reactivity with endogenous expressed SLA-DR in a variety of cell lines and primary swine antigen-presenting cells (PAMs, PBMC and BM-DCs). Moreover, the level of SLA-DR expression after PRRSV infection were evaluated by our homemade Mab and a commercial anti-SLA-DR antibody. Based on our result, the protein level of SLA-DRα expression is increased after PRRSV infection in DC, while the mRNA of both SLA-DRα and SLA-DRß were significantly inhibited by PRRSV replication. In conclusion, we successfully developed a MAb reactive with endogenous SLA-DR in western blotting, and this MAb could be a useful tool for further research and analysis. Moreover, the inconsistency of SLA-DR expression between protein and mRNA levels may suggest a novel role of DC played during the immune response after PRRSV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Células Dendríticas/metabolismo , Cadeias alfa de HLA-DR/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Animais , Western Blotting , Medula Óssea/imunologia , Medula Óssea/metabolismo , Linhagem Celular , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Células HEK293 , Cadeias alfa de HLA-DR/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Proteínas Recombinantes , Suínos/imunologia
13.
Nat Protoc ; 14(6): 1734-1755, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31053798

RESUMO

R-loops are prevalent three-stranded non-B DNA structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops are implicated in various basic nuclear processes, such as class-switch recombination, transcription termination and chromatin patterning. Perturbations in R-loop metabolism have been linked to genomic instability and have been implicated in human disorders, including cancer. As a consequence, the accurate mapping of these structures has been of increasing interest in recent years. Here, we describe two related immunoprecipitation-based methods for mapping R-loop structures: basic DRIP-seq (DNA-RNA immunoprecipitation followed by high-throughput DNA sequencing), an easy, robust, but resolution-limited technique; and DRIPc-seq (DNA-RNA immunoprecipitation followed by cDNA conversion coupled to high-throughput sequencing), a high-resolution and strand-specific iteration of the method that permits accurate R-loop mapping genome wide. Briefly, after gentle DNA extraction and restriction digestion with a cocktail of enzymes, R-loop structures are immunoprecipitated with the anti-RNA-DNA hybrid S9.6 antibody. Compared with DRIP-seq, in which the immunoprecipitated DNA is directly sequenced, DRIPc-seq permits the recovery of the RNA moiety of R-loops, and these RNA strands are subjected to strand-specific RNA sequencing (RNA-seq) analysis. DRIPc-seq can be performed in 5 d and can be applied to any cell type, provided sufficient starting material can be collected. Accurately mapping R-loop distribution in various cell lines and under varied conditions is essential to understanding the formation, roles and dynamic resolution of these important structures.


Assuntos
DNA/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , RNA/análise , Animais , Anticorpos/química , Anticorpos Monoclonais/química , DNA/genética , Humanos , Camundongos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , RNA/genética
14.
Biomed Res Int ; 2019: 1856180, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019965

RESUMO

Objective: This study aimed to clarify the clinical features, the serum level of autoantibodies, and cytokine of myositis patients with anti-EJ antibody, which targets glycyl tRNA-synthetase (GlyRS). Methods: Sera of 236 Chinese patients with myositis were screened for anti-EJ by a novel immunoprecipitation assay of flag-tagged GlyRS. Anti-EJ positive patients are evaluated for the clinical features and cytokine profile. Results: The sera from 4 of 236 adult myositis patients were found to carry the anti-EJ using established novel immunoprecipitation assay and immunoblotting. The prevalence of anti-EJ in our cohorts is about 1.7%. The decline of anti-EJ level was detected in two patients during disease remission. Interstitial lung disease and muscle weakness, but not skin involvement, are common clinical features of anti-EJ positive patients. Moreover, using a cytokine profile analyses, we found that the serum levels of IP-10, IL-6, MCP-1, and VEGF were significantly elevated in patients with anti-EJ and gradually decreased during disease remission of two patients, whereas IL-8 level was obviously reduced in these patients. Conclusion: The novel immunoprecipitation assay is suitable to detect and monitor the levels of anti-EJ autoantibody. The serum levels of anti-EJ, IP-10, IL-6, MCP-1, and VEGF may be related to disease activity in myositis patients with anti-EJ antibodies.


Assuntos
Autoanticorpos/sangue , Citocinas/sangue , Miosite/sangue , Adulto , Autoanticorpos/imunologia , Citocinas/imunologia , Feminino , Humanos , Imunoprecipitação/métodos , Doenças Pulmonares Intersticiais/sangue , Doenças Pulmonares Intersticiais/imunologia , Masculino , Pessoa de Meia-Idade , Miosite/imunologia
15.
Int J Mol Med ; 43(6): 2267-2278, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31017262

RESUMO

Among a number of mRNA modifications, N6­methyladenosine (m6A) modification is the most common type in eukaryotes and nuclear­replicating viruses. m6A has a significant role in numerous cancer types, including leukemia, brain tumors, liver cancer, breast cancer and lung cancer. Although m6A methyltransferases are essential during RNA modifications, the biological functions of m6A and the underlying mechanisms remain to be fully elucidated, predominantly due to the limited detection methods for m6A. In the present review, the currently available m6A detection methods and the respective scope of their applications are presented to facilitate the further investigation of the roles of m6A in biological process.


Assuntos
Adenosina/análogos & derivados , RNA/química , Adenosina/análise , Adenosina/genética , Animais , Técnicas Biossensoriais/métodos , Northern Blotting/métodos , Cromatografia Líquida de Alta Pressão/métodos , Técnicas Eletroquímicas/métodos , Humanos , Immunoblotting/métodos , Imunoprecipitação/métodos , Metilação , Neoplasias/genética , RNA/genética , Análise de Sequência de RNA/métodos
16.
Methods Mol Biol ; 1981: 149-162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016653

RESUMO

Necroptosis is emerging as a critical pathogenic mechanism in several liver diseases, including cholestatic disorders. Necroptosis was recently described as a novel cell death subroutine, activated downstream of death receptor stimulation and dependent on receptor-interacting serine/threonine-protein kinase 3 activity and mixed lineage kinase domain-like oligomerization and translocation to cell membrane. Here, we describe a combination of methods to evaluate necroptosis triggering in in vitro and in vivo models of cholestasis. Particularly, we detail alternative protocols to isolate total and soluble/insoluble protein extracts from tissues and cell cultures, as well as in vitro receptor-interacting serine/threonine-protein kinase 3 kinase activity assays, and subsequent Western blot analysis.


Assuntos
Colestase/metabolismo , Colestase/patologia , Animais , Apoptose/fisiologia , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Necrose/metabolismo , Necrose/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/fisiologia
17.
Methods Mol Biol ; 1933: 279-288, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945192

RESUMO

Recent advances in next-generation sequencing have revealed that majority of the plant genome is transcribed into long noncoding RNA (lncRNA). Many lncRNAs function by interacting with proteins and forming regulatory complexes. RNA-protein interactions are vital in controlling core cellular processes like transcription and translation. Therefore, identifying proteins that interact with lncRNAs is the first step to deciphering lncRNA functions. Here, we describe an RNA-protein pull-down assay, which enables the identification of proteins that interact with an RNA under study. As an example, we describe pull-down of proteins interacting with lncRNA ELENA1, which promotes the enrichment of MED19a on PR1 promoter to activate PR1 expression.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Imunoprecipitação/métodos , Extratos Vegetais/metabolismo , RNA Longo não Codificante/metabolismo , RNA de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Extratos Vegetais/genética , RNA Longo não Codificante/genética , RNA de Plantas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma
18.
Methods Mol Biol ; 1933: 289-295, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945193

RESUMO

Long noncoding RNAs (lncRNAs) play important roles in several processes including control of gene expression. These RNAs function through binding to histone-modifying complexes and transcriptional machinery including transcription factor, mediator, and RNA polymerase II. We present methods for the discovery and characterization of lncRNAs. RNA immunoprecipitation (RIP) is a modified version of chromatin immunoprecipitation (ChIP), and it is now generally used in lncRNA study. The method allows for testing of lncRNA-protein interactions in vivo. RIP assay facilitates the identification of consensus sequences of preferred binding site for the RNA-binding protein under study, and identification of the binding sites can provide valuable information on the possible mechanism by which the RNA-binding protein functions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Imunoprecipitação da Cromatina/métodos , Imunoprecipitação/métodos , RNA Longo não Codificante/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Longo não Codificante/genética , RNA de Plantas/genética , Proteínas de Ligação a RNA/genética
19.
Methods Mol Biol ; 1933: 389-394, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945199

RESUMO

Transcriptome-wide mapping RNA modification is crucial to understand the distribution and function of RNA modifications. Here, we describe a protocol to transcriptome-wide mapping 5-methylcytosine (m5C) in plant, by a RNA immunoprecipitation followed by deep sequencing (m5C-RIP-seq) approach. The procedure includes RNA extraction, fragmentation, RNA immunoprecipitation, and library construction.


Assuntos
5-Metilcitosina/química , Arabidopsis/genética , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , RNA de Plantas/genética , Transcriptoma , Biologia Computacional , RNA de Plantas/química , Análise de Sequência de RNA/métodos
20.
Vet Res ; 50(1): 28, 2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31029162

RESUMO

Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus that causes diarrhea in pigs and is associated with high morbidity and mortality in sucking piglets. S1 is one of two protein domains in the spike (S) glycoprotein and is responsible for enteric tropism, sialic acid recognition, and host receptor binding. Although there has been extensive research on the S1 protein of TGEV, little is known about the intracellular role of TGEV-S1. In the present study, we used yeast two-hybrid screening of a cDNA library from porcine intestinal cells to identify proteins that interact with TGEV-S1. Among 120 positive clones from the library, 12 intracellular proteins were identified after sequencing and a BLAST search. These intracellular proteins are involved in protein synthesis and degradation, biological signal transduction, and negative control of signaling pathways. Using a glutathione-S-transferase (GST) pulldown assay and Co-IP, we found that UBXN1 interacts with the S1 protein. Here, we observed that TGEV infection led to increased UBXN1 expression levels during the late phase of infection in IPEC-J2 cells. Inhibition of UBXN1 in IPEC-J2 cells via siRNA interference significantly decreased the viral titer and downregulated the expression of S1. UBXN1 overexpression significantly increased the viral copy number. Additionally, we provided data suggesting that UBXN1 negatively regulates IFN-ß expression after TGEV infection. Finally, our research indicated that UBXN1 plays a vital role in the process of TGEV infection, making it a candidate target for the development of a novel antiviral method.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Vírus da Gastroenterite Transmissível/fisiologia , Proteínas Virais/fisiologia , Replicação Viral , Western Blotting , Ensaio de Imunoadsorção Enzimática , Imunoprecipitação , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-Híbrido
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA