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1.
Rapid Commun Mass Spectrom ; 34(2): e8573, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31484223

RESUMO

RATIONALE: Lys-N, also known as lysine-specific metalloendopeptidase, functions as the "sister" enzyme of lysyl endopeptidase (Lys-C) in proteomic research. Its digestion specificity at the N-terminal lysine residue makes it a very useful tool in proteomics analysis, especially in mass spectrometry (MS)-based de novo sequencing of proteins. METHODS: Here we present a complete production process of highly purified Lys-N from dry fruit of Grifola frondosa (maitake mushroom). The purification process includes one step of microfiltration plus one step of UF/DF (ultrafiltration used in tandem with a diafiltration method) recovery and four steps of chromatographic purification. RESULTS: The overall yield of the process was approximately 6.7 mg Lys-N protein/kg dry fruit of G. frondosa. The assay data demonstrated that the purified Lys-N exhibited high enzymatic activity and specificity. CONCLUSIONS: The novel production process provides for the first time the extraction of Lys-N from dry fruit of G. frondosa. The process is also stable and scalable, and provides an economic way of producing the enzyme in large quantities for MS-based proteomics and other biological studies.

2.
J Proteomics ; 213: 103614, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31846764

RESUMO

Lysine methylation is a widespread protein post-translational modification showing essentialities in versatile cellular process. EZH2, a methyltransferase specifically trimethylates the lysine 27 of histone H3 and its aberrance in several cancers promotes the development of its inhibitors against hematological tumors. In this study, we presented a deep exploration of lysine mono-, di- and trimethylomes in EZH2 wild-type and Y641 mutant lymphoma cell lines. Our results showed that several substrates were modified in different methylation levels. Moreover, these methylated lysine residues could also undergo other types of PTMs. Combined with the differences proved in protein expression, lysine acetylation, lysine ubiquitylation and protein N-termianl acetylation level, our study underlined the substrate specificity of lysine methylation and its crosstalk with other types of PTMs. Totally, our study raised new insights into the global cellular methylation features in hematological cell lines, which provided further inspects into the distribution and function of lysine methylation. SIGNIFICANCE: Our study showed the global landscape of mono-, di- and trimethylomes in the EZH2-aberrant DLBCL cell lines, revealing the molecular characteristics of lysine methylation. Combined with the protein abundance and potential crosstalk among different types of PTMs, our study raised new insights into the global cellular methylation features in hematological tumors and provided further inspects into the distribution and function of lysine methylation.

3.
EMBO J ; 38(18): e100948, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31418899

RESUMO

As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c-di-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.


Assuntos
GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Sirtuínas/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , GMP Cíclico/metabolismo , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Análise Serial de Proteínas/métodos , Proteômica/métodos , Sistemas do Segundo Mensageiro
4.
Cell ; 175(1): 186-199.e19, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30220457

RESUMO

Mutations or aberrant upregulation of EZH2 occur frequently in human cancers, yet clinical benefits of EZH2 inhibitor (EZH2i) remain unsatisfactory and limited to certain hematological malignancies. We profile global posttranslational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2i. We report here oncogenic transcriptional reprogramming mediated by MLL1's interaction with the p300/CBP complex, which directs H3K27me loss to reciprocal H3K27ac gain and restricts EZH2i response. Concurrent inhibition of H3K27me and H3K27ac results in transcriptional repression and MAPK pathway dependency in cancer subsets. In preclinical models encompassing a broad spectrum of EZH2-aberrant solid tumors, a combination of EZH2 and BRD4 inhibitors, or a triple-combination including MAPK inhibition display robust efficacy with very tolerable toxicity. Our results suggest an attractive precision treatment strategy for EZH2-aberrant tumors on the basis of tumor-intrinsic MLL1 expression and concurrent inhibition of epigenetic crosstalk and feedback MAPK activation.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Histona-Lisina N-Metiltransferase/fisiologia , Proteína de Leucina Linfoide-Mieloide/fisiologia , Animais , Carcinogênese/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Epigênese Genética/genética , Epigenômica/métodos , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Código das Histonas/efeitos dos fármacos , Código das Histonas/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histonas/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Ativação Transcricional , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Fatores de Transcrição de p300-CBP/fisiologia
5.
Guang Pu Xue Yu Guang Pu Fen Xi ; 27(3): 589-91, 2007 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-17554929

RESUMO

A method was proposed for the determination of trace copper and lead in beer with flame atomic absorption spectrometry after preconcentration of copper and lead by rapid coprecipitation technique with 8-oxyquinoline-Mg(II) using manganese as an internal standard at pH 9. The standard addition recovery of lead is between 97.6%-103.0%. The detection limit is 6.28 x 10(-3) microg x mL(-1) for copper and 2.26 x 10(-2) microg x mL(-1) for lead when the sample volume is 100 mL. The effect of matrix can be overcome by the method and the results are satisfying. The method proposed here is rapid and has good reproducibility.


Assuntos
Cerveja/análise , Cobre/isolamento & purificação , Chumbo/isolamento & purificação , Oxiquinolina/química , Espectrofotometria Atômica/métodos , Adsorção , Precipitação Química , Cobre/química , Chumbo/química , Reprodutibilidade dos Testes , Fatores de Tempo
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