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.

There was an error published in J. Cell Sci. 129, 2213-2223.In Fig. 4A, a statement that duplicate α-tubulin blots are shown for Mcl-1 and Bok, and for caspase-3 and Bax, because the same membranes were probed for these proteins, was inadvertently omitted from the legend.The authors apologise to the readers for any confusion that this error might have caused. 3054

There was an error published in J. Cell Sci. 129, 2213-2223.In Fig. 4A, a statement that duplicate α-tubulin blots are shown for Mcl-1 and Bok, and for caspase-3 and Bax, because the same membranes were probed for these proteins, was inadvertently omitted from the legend.The authors apologise to the readers for any confusion that this error might have caused.

Central to intrinsic apoptosis signaling is the release of cytochrome c from mitochondria, which depends on the pro-apoptotic effector proteins Bax, Bak or Bok. These pore-forming effector proteins share four Bcl-2 homology (BH) domains, a functionally essential and conserved sequence of hydrophobic amino acids in their BH3-domain and a C-terminal transmembrane-domain whose specific function remains rather unknown. To elucidate the molecular basis of Bok-mediated apoptosis we analyzed apoptosis induction by transmembrane-domain deficient BokΔTM compared to the respective Bax and Bak proteins and proteins in which the first leucine in the BH3-stretch was mutated to glutamic acid. We show that deletion of the C-terminal transmembrane-domain reduces the pro-apoptotic function of each protein. Mutation of the first leucine in the BH3-domain (L78E) blocks activity of Bak, while mutation of the homologue residues in Bax or Bok (L63E and L70E respectively) does not affect apoptosis induction. Unexpectedly, combined mutation of the BH3-domain and deletion of the transmembrane-domain enhances the pro-apoptotic activity of Bok(L70E)ΔTM by abolishing the interaction with anti-apoptotic proteins, especially the primary Bok-inhibitory protein Mcl-1. These results therefore suggest a specific contribution of the transmembrane-domain to the pro-apoptotic function and interaction of Bok.

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