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1.
Methods Mol Biol ; 1983: 79-106, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31087294

RESUMO

Posttranslational modifications of proteins control many complex biological processes, including genome expression, chromatin dynamics, metabolism, and cell division through a language of chemical modifications. Improvements in mass spectrometry-based proteomics have demonstrated protein acetylation is a widespread and dynamic modification in the cell; however, many questions remain on the regulation and downstream effects, and an assessment of the overall acetylation stoichiometry is needed. In this chapter, we describe the determination of acetylation stoichiometry using data-independent acquisition mass spectrometry to expand the number of acetylation sites quantified. However, the increased depth of data-independent acquisition is limited by the spectral library used to deconvolute fragmentation spectra. We describe a powerful approach of subcellular fractionation in conjunction with offline prefractionation to increase the depth of the spectral library. This deep interrogation of subcellular compartments provides essential insights into the compartment-specific regulation and downstream functions of protein acetylation.


Assuntos
Lisina/metabolismo , Proteínas/metabolismo , Acetilação , Técnicas de Cultura de Células , Fracionamento Celular , Cromatografia Líquida , Cromatografia de Fase Reversa , Interpretação Estatística de Dados , Concentração de Íons de Hidrogênio , Lisina/química , Espectrometria de Massas/métodos , Mitocôndrias/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteômica/métodos , Espectrometria de Massas em Tandem
2.
ACS Chem Biol ; 12(11): 2804-2814, 2017 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-28949514

RESUMO

There is a tremendous need for novel strategies aimed at directly assessing activities of histone modifiers to probe epigenetic determinants associated with disease progression. Here, we developed a high-throughput peptide microarray assay to identify altered histone lysine (de)acetylation activity in prostate cancer (PCa). This microarray-based activity assay revealed up-regulated histone acetyltransferase (HAT) activity against specific histone H3 sites in a castrate-resistant (CR) PCa cell line compared to its hormone-sensitive (HS) isogenic counterpart. NAD+-dependent deacetylation assays revealed down-regulated sirtuin activity in validated CR lines. Levels of acetyltransferases GCN5, PCAF, CBP, and p300 were unchanged between matched HS and CR cell lines. However, autoacetylation of p300 at K1499, a modification known to enhance HAT activity and a target of deacetylation by SIRT2, was highly elevated in CR cells, while SIRT2 protein level was reduced in CR cells. Interrogation of HS and matched CR xenograft lines reveals that H3K18 hyperacetylation, increased p300 activity, and decreased SIRT2 expression are associated with progression to CR in 8/12 (66%). Tissue microarray analysis revealed that hyperacetylation of H3K18 is a feature of CRPC. Inhibition of p300 results in lower H3K18ac levels and increased expression of androgen receptors. Thus, a novel histone array identifies altered enzyme activities during the progression to CRPC and may be utilized in a personalized medicine approach. Reduced SIRT2 expression and increased p300 activity lead to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC.


Assuntos
Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Neoplasias da Próstata/enzimologia , Sirtuínas/metabolismo , Acetilação , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Humanos , Masculino , Camundongos , Orquiectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Análise Serial de Proteínas , Sirtuína 2/metabolismo , Xenopus
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