A miniaturized readout strategy for endogenous histone deacetylase activity

Jost, Jan Oliver; Hanswillemenke, Alfred; Schwarzer, Dirk

doi:10.1039/c5mb00326a

Cited by 5 publications

(6 citation statements)

References 26 publications

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“…[12] In case of HDAC8, the recombinant enzyme shows astrong preference for p53-K382ac-derived substrates,but the pull-down experiments with the p53 probes do not support amajor role of this HDAC in the deacetylation of K382ac. In order to validate whether the pull-down experiments with the newly established HDAC probes enable proper predictions of deacetylase substrate selectivity,westudied the catalytic properties of selected HDACsincellular lysates.Wehave previously shown that global deacetylase activity in cellular lysates can be monitored with synthetic acetylated peptides by MALDI-TOFM S. [13] We synthesized ap 53-derived peptide substrate with acetylated K382 that was readily deacetylated by endogenous HDACsf rom cell lysates ( Figure S7). An isotope-labeled reference peptide was used to quantify the amount of formed deacetylated product.…”

Section: Angewandte Chemiementioning

confidence: 99%

“…In order to validate whether the pull‐down experiments with the newly established HDAC probes enable proper predictions of deacetylase substrate selectivity, we studied the catalytic properties of selected HDACs in cellular lysates. We have previously shown that global deacetylase activity in cellular lysates can be monitored with synthetic acetylated peptides by MALDI‐TOF MS . We synthesized a p53‐derived peptide substrate with acetylated K382 that was readily deacetylated by endogenous HDACs from cell lysates (Figure S7).…”

Section: Figurementioning

confidence: 99%

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Interrogating Substrate Selectivity and Composition of Endogenous Histone Deacetylase Complexes with Chemical Probes

et al. 2015

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Histone deacetylases (HDACs) regulate the function and activity of numerous cellular proteins by removing acetylation marks from regulatory lysine residues. We have developed peptide-based HDAC probes that contain hydroxamate amino acids of various lengths to replace modified lysine residues in the context of known acetylation sites. The interaction profiles of all human HDACs were studied with three sets of probes, which derived from different acetylation sites, and sequence context was found to have a strong impact on substrate recognition and composition of HDAC complexes. By investigating K382 acetylation of the tumor suppressor p53 as an example, we further demonstrate that the interaction profiles reflect the catalytic activities of respective HDACs. These results underline the utility of the newly established probes for deciphering not only activity, but also substrate selectivity and composition of endogenous HDAC complexes, which can hardly be achieved otherwise.

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Section: Angewandte Chemiementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Interrogating Substrate Selectivity and Composition of Endogenous Histone Deacetylase Complexes with Chemical Probes

et al. 2015

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“…Um zu überprüfen, ob die Pulldown‐Versuche mit den neu etablierten HDAC‐Sonden ein korrektes Vorhersagen der Substratselektivität der Deacetylase ermöglichen, untersuchten wir die katalytischen Eigenschaften von ausgewählten HDACs in Zelllysaten. Wie zuvor gezeigt, lässt sich die globale Deacetylase‐Aktivität in Zelllysaten mit synthetischen acetylierten Peptiden mittels MALDI‐TOF‐MS quantitativ bestimmen . Ein p53‐Peptid‐Substrat mit acetyliertem K382 wurde im Folgenden durch endogene HDACs aus Zelllysaten deacetyliert und die Produktbildung unter Zuhilfenahme eines isotopenmarkierten Referenzpeptids quantitativ untersucht (Abbildung S7).…”

Section: Figureunclassified

Untersuchung der Substratselektivität und Zusammensetzung endogener Histondeacetylase‐Komplexe durch chemische Sonden

et al. 2015

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Die HDAC-Komplexe sind generell wenig verstanden und der gegenwärtige wissenschaftliche Fokus liegt auf der Erforschung,i nwiefern sie Aktivität und Selektivität der Deacetylase modulieren.[5] Eine aktuelle Herangehensweise zur Erforschung von Substraten der HDAC-Komplexe ist die Bestimmung der Lysin-Acetylome von Zellen oder Organismen, die mit HDAC-Inhibitoren behandelt wurden.[6] Zusätzlich sind aktivitätsbasierte Sonden und immobilisierte HDAC-Inhibitoren vielversprechend für die Untersuchung von HDAC-Aktivität in Zelllysaten. [7] Im Folgenden stellen wir die Synthese von peptidbasierten Sonden vor, um Substratselektivität und Zusammensetzung von endogenen HDAC-Komplexen von Säugetieren zu untersuchen. Diese Sonden beinhalten synthetisierte Aminosäure-Bausteine,d eren funktionelle Gruppe sich von bekannten HDAC-Inhibitoren ableitet. Wirh aben dabei auf Hydroxamsäuren zurückgegriffen, die eine Klasse von HDAC-Inhibitoren bilden und in der Lage sind, das katalytische Zn 2+ -Ion der HDACsm it nanomolarer Affinität zu chelatisieren. [2, 7a] Die synthetisierten Hydroxamat-Bausteine ersetzen acetylierte Lysine in Peptiden, die von bekannten Acetylierungsstellen abgeleitet wurden. Der Abstand zwischen Hydroxamat-Gruppe und Peptid-Rückgrat schien kritisch, weshalb wir die Kettenlängen der Hydroxamat-Bausteine zwischen fünf (2-Aminosuberinsäure-w-hydroxamatAsuHd), vier (2-Aminoheptandisäure-e-hydroxamatApmHd), drei (2-Aminoadipinsäure-d-hydroxamatAadHd) und zwei (Glutaminsäure-g-hydroxamat -G luHd) Methylengruppen variiert haben (Abbildung 1).[8] Die AsuHd-(1)u nd ApmHd-Bausteine (2)w urden über bereits

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“…HDAC‐catalysis requires metal ions for water activation in order to cleave the N ε‐amide bond of acetyllysine substrates. Although there is evidence that recombinant HDAC8 can utilize Co 2+ and Fe 2+ , in most cases Zn 2+ is considered as the catalytic metal ion of HDACs . Based on this, hydroxamic acid compounds that chelate the catalytic Zn 2+ ion are among the most potent HDAC inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Probing the structure–activity relationship of endogenous histone deacetylase complexes with immobilized peptide‐inhibitors

Sindlinger

Bierlmeier

Geiger

et al. 2016

Journal of Peptide Science

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Histone deacetylases (HDACs) are key regulators of numerous cellular proteins by removing acetylation marks from modified lysine residues. Peptide-based HDAC probes containing α-aminosuberic acid ω-hydroxamate have been established as useful tools for investigating substrate selectivity and composition of endogenous HDAC complexes in cellular lysates. Here we report a structure-activity study of potential HDAC-probes containing derivatives of the hydroxamate moieties. While most of these probes did not recruit significant amounts of endogenous HDACs from cellular lysates, peptides containing Nε-acetyl-Nε-hydroxy-L-lysine served as HDAC probe. The recruitment efficiency varied between HDACs and was generally lower than that of α-aminosuberic acid ω-hydroxamate probes, but showed a similar global interaction profile. These findings indicate that Nε-acetyl-Nε-hydroxy-L-lysine might be a useful tool for investigations on HDAC complexes and the development of HDAC inhibitors. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

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A miniaturized readout strategy for endogenous histone deacetylase activity

Cited by 5 publications

References 26 publications

Interrogating Substrate Selectivity and Composition of Endogenous Histone Deacetylase Complexes with Chemical Probes

Interrogating Substrate Selectivity and Composition of Endogenous Histone Deacetylase Complexes with Chemical Probes

Untersuchung der Substratselektivität und Zusammensetzung endogener Histondeacetylase‐Komplexe durch chemische Sonden

Probing the structure–activity relationship of endogenous histone deacetylase complexes with immobilized peptide‐inhibitors

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