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1.
Protein Expr Purif ; 141: 1-6, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28843507

RESUMO

Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation. Characterization of KDAC activity and substrate identification is complicated by inconsistent activity of prepared enzyme and a range of multi-step purifications. We describe a simplified protocol based on two-step affinity chromatography. The purification method is appropriate for use regardless of expression host, and we demonstrate purification of several representative members of the KDAC family as well as a selection of mutated variants. The purified proteins are highly active and consistent across preparations.


Assuntos
Cobalto/metabolismo , Histona Desacetilases/isolamento & purificação , Histona Desacetilases/metabolismo , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Animais , Dicroísmo Circular , Cobalto/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Histona Desacetilases/química , Histona Desacetilases/genética , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Células Sf9/metabolismo
2.
Biochemistry ; 56(34): 4549-4558, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28749131

RESUMO

Lysine deacetylases (KDACs) are enzymes that reverse the post-translational modification of lysine acetylation. Thousands of potential substrates, acetylated protein sequences, have been identified in mammalian cells. Properly regulated acetylation and deacetylation have been linked to many biological processes, while aberrant KDAC activity has also been linked to numerous diseases. Commercially available peptide substrates that are conjugated to fluorescent dye molecules, such as 7-amino-4-methylcoumarin (AMC), are commonly used to monitor deacetylation in studies addressing both substrate specificity and small molecule modulators of activity. Here, we have compared the activity of several KDACs, representing all major classes of KDACs, with substrates in the presence and absence of AMC as well as peptides for which tryptophan has been substituted for AMC. Our results unequivocally demonstrate that AMC has a significant effect on activity for all KDACs tested. Furthermore, in neither the nature of the effect nor the magnitude is consistent across KDACs, making it impossible to predict the effect of AMC on a particular enzyme-substrate pair. AMC did not affect acetyllysine preference in a multiply acetylated substrate. In contrast, AMC significantly enhanced KDAC6 substrate affinity, greatly reduced Sirt1 activity, eliminated the substrate sequence specificity of KDAC4, and had no consistent effect with KDAC8 substrates. These results indicate that profiling of KDAC activity with labeled peptides is unlikely to produce biologically relevant data.


Assuntos
Cumarínicos/química , Histona Desacetilases/química , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Especificidade por Substrato
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