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1.
Nat Methods ; 17(10): 985-988, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32994567

RESUMO

Thorough quality assessment of novel interactions identified by proteome-wide cross-linking mass spectrometry (XL-MS) studies is critical. Almost all current XL-MS studies have validated cross-links against known three-dimensional structures of representative protein complexes. Here, we provide theoretical and experimental evidence demonstrating that this approach can drastically underestimate error rates for proteome-wide XL-MS datasets, and propose a comprehensive set of four data-quality metrics to address this issue.

2.
Proc Natl Acad Sci U S A ; 117(21): 11836-11842, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32398372

RESUMO

Systematic mappings of protein interactome networks have provided invaluable functional information for numerous model organisms. Here we develop PCR-mediated Linkage of barcoded Adapters To nucleic acid Elements for sequencing (PLATE-seq) that serves as a general tool to rapidly sequence thousands of DNA elements. We validate its utility by generating the ORFeome for Oryza sativa covering 2,300 genes and constructing a high-quality protein-protein interactome map consisting of 322 interactions between 289 proteins, expanding the known interactions in rice by roughly 50%. Our work paves the way for high-throughput profiling of protein-protein interactions in a wide range of organisms.


Assuntos
Fases de Leitura Aberta/genética , Oryza/genética , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Análise de Sequência de DNA/métodos , Biologia Computacional/métodos , DNA de Plantas/genética , Bases de Dados Genéticas , Genoma de Planta/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos
3.
Mol Cell Proteomics ; 19(3): 554-568, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31839598

RESUMO

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry (XL-MS) has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. The inception of MS-cleavable cross-linkers enabled the MS2-MS3 XL-MS acquisition strategy that provides cross-link information from both MS2 and MS3 level. However, the current cross-link search algorithm available for MS2-MS3 strategy follows a "MS2-centric" approach and suffers from a high rate of mis-identified cross-links. We demonstrate the problem using two new quality assessment metrics ["fraction of mis-identifications" (FMI) and "fraction of interprotein cross-links from known interactions" (FKI)]. We then address this problem, by designing a novel "MS3-centric" approach for cross-link identification and implementing it as a search engine named MaXLinker. MaXLinker outperforms the currently popular search engine with a lower mis-identification rate, and higher sensitivity and specificity. Moreover, we performed human proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we identified a comprehensive set of 9319 unique cross-links at 1% false discovery rate, comprising 8051 intraprotein and 1268 interprotein cross-links. Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinker's robust performance.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteômica/métodos , Humanos , Células K562 , Espectrometria de Massas , Peptídeos/metabolismo , Proteoma , Sensibilidade e Especificidade
4.
Eur J Med Chem ; 147: 130-149, 2018 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-29427906

RESUMO

The ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) was recently shown to promote mineralization of the aortic valve, hence, its inhibition represents a significant target. A quinazoline-4-piperidine sulfamide compound (QPS1) has been described as a specific and non-competitive inhibitor of NPP1. We report herein the synthesis and in vitro inhibition studies of novel quinazoline-4-piperidine sulfamide analogues using QPS1 as the lead compound. Of the 26 derivatives prepared, four compounds were found to have Ki < 105 nM against human NPP1.


Assuntos
Amidas/farmacologia , Inibidores Enzimáticos/farmacologia , Piperidinas/farmacologia , Pirofosfatases/antagonistas & inibidores , Quinazolinas/farmacologia , Amidas/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Diester Fosfórico Hidrolases/metabolismo , Piperidinas/química , Pirofosfatases/metabolismo , Quinazolinas/química , Relação Estrutura-Atividade
5.
Br J Pharmacol ; 172(16): 4189-99, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26031197

RESUMO

BACKGROUND AND PURPOSE: Ectonucleotide pyrophosphatase/PDE1 (NPP1) is an ectoenzyme, which plays a role in several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack potency and specificity. Quinazoline-4-piperidine sulfamides (QPS) have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. EXPERIMENTAL APPROACH: In the present series of experiments, we have evaluated the efficacy of two derivatives, QPS1-2, in inhibiting human NPP1, and we have evaluated the effect of the most potent derivative (QPS1) on other ectonucleotidases as well as on the ability of this compound to prevent phosphate-induced mineralization of human primary aortic valve interstitial cells (VICs). KEY RESULTS: The QPS1 derivative is a potent (Ki 59.3 ± 5.4 nM) and selective non-competitive inhibitor of human NPP1. Moreover, QPS1 also significantly inhibited the K121Q NPP1 gene variant (Ki 59.2 ± 14.5 nM), which is prevalent in the general population. QPS1 did not significantly alter the activity of other nucleotide metabolizing ectoenzymes expressed at the cell surface, namely NPP3, NTPDases (1-3), ecto-5'-nucleotidase and ALP. Importantly, QPS1 in the low micromolar range (≤10 µM) prevented phosphate-induced mineralization of VICs and lowered the rise of osteogenic genes as expected for NPP1 inhibition. CONCLUSIONS AND IMPLICATIONS: We have provided evidence that QPS1 is a potent and selective non-competitive inhibitor of NPP1 and that it prevented pathological mineralization in a cellular model.


Assuntos
Pirofosfatases/antagonistas & inibidores , Quinazolinas/farmacologia , Adulto , Animais , Valva Aórtica/citologia , Apoptose/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Diester Fosfórico Hidrolases
6.
J Mol Cell Cardiol ; 82: 104-15, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25771146

RESUMO

AIMS: Calcific aortic valve stenosis (CAVS) is the most common heart valve disease. In the present work we sought to determine the reversibility of mineralization in the aortic valve. METHODS AND RESULTS: By using in vitro analyses we found that valve interstitial cells (VICs) have the ability to resorb minerals. We documented that agonist of P2Y2 receptor (P2Y2R) promoted the expression of carbonic anhydrase XII (CAXII) at the cell membrane of VICs, whereby minerals are resorbed. P2Y2R-mediated mineral resorption was corroborated by using mouse VICs isolated from wild type and P2Y2R(-/-) mice. Measurements of extracellular pH (pHe) by using core-shell nanosensors revealed that P2Y2R-mediated CAXII export to the cell membrane led to an acidification of extracellular space, whereby minerals are resorbed. In vivo, we next treated LDLR(-/-)/ApoB(100/100)/IGF2 mice, which had developed CAVS under a high-fat/high-sucrose diet for 8 months, with 2-thioUTP (a P2Y2R agonist) or saline for the next 2 months. The administration of 2-thioUTP (2mg/kg/day i.p.) reduced the mineral volume in the aortic valve measured with serial microCT analyses, which improved hemodynamics and reduced left ventricular hypertrophy (LVH). Examination of leaflets at necropsy confirmed a lower level of mineralization and fibrosis along with higher levels of CAXII in mice under 2-thioUTP. In another series of experiment, the administration of acetazolamide (a CA inhibitor) prevented the acidification of leaflets and the regression of CAVS induced by 2-thioUTP in LDLR(-/-)/ApoB(100/100)/IGF2 mice. CONCLUSION: P2Y2R-mediated expression of CAXII by VICs acidifies the extracellular space and promotes the regression of CAVS.


Assuntos
Estenose da Valva Aórtica/etiologia , Estenose da Valva Aórtica/metabolismo , Calcinose/complicações , Calcinose/metabolismo , Anidrases Carbônicas/metabolismo , Valvas Cardíacas/metabolismo , Animais , Estenose da Valva Aórtica/diagnóstico , Estenose da Valva Aórtica/tratamento farmacológico , Calcinose/patologia , Modelos Animais de Doenças , Espaço Extracelular/metabolismo , Valvas Cardíacas/patologia , Masculino , Camundongos , Camundongos Transgênicos , Minerais/metabolismo , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y2/metabolismo
7.
Clin Respir J ; 8(1): 108-15, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23902466

RESUMO

BACKGROUND: The T1 (rs2280091), S1 (rs3918396) and S2 (rs528557) polymorphisms in a disintegrin and metalloprotease (ADAM33) gene has been implicated in susceptibility of chronic obstructive pulmonary disease (COPD). But, a number of studies have reported inconclusive results. The aim of this study is to investigate the relationship between T1 (rs2280091), S1 (rs3918396) and S2 (rs528557) polymorphisms in ADAM33 gene and COPD risk by meta-analysis. METHODS: We searched PubMed database, Embase database, Chinese National Knowledge Infrastructure database and Wanfang database, covering all studies till September 5, 2012. Statistical analysis was performed using software METAGEN (STATA 12.0) and Revman5.0. RESULTS: A total of 2139 COPD cases and 3765 controls in 10 case-control studies were included in this study. The results showed that S2 (rs528557) and T1 (rs2280091) polymorphisms did not result in an increased or a decreased risk of COPD. The analysis described in this report demonstrated that S1 (rs3918396) polymorphism (GG + AG vs AA) was significantly associated with the total and Asian. Odds ratio (OR)total = 1.27 [95% confidence interval (CI) 1.03-1.56, P = 0.03], ORAsian = 1.44 (95% CI 1.13-1.83, P = 0.003) but not with Caucasians. CONCLUSIONS: This meta-analysis suggested that S1 (rs3918396) polymorphism of ADAM33 is associated with increased risk of COPD in Asian (China) but not in Caucasians. Future studies are needed to validate our conclusions.


Assuntos
Proteínas ADAM/genética , Grupo com Ancestrais do Continente Asiático/genética , Grupo com Ancestrais do Continente Europeu/genética , Polimorfismo de Nucleotídeo Único/genética , Doença Pulmonar Obstrutiva Crônica/genética , Humanos , Medição de Risco
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