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

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.

A Multiplexed NMR-Reporter Approach to Measure Cellular Kinase and Phosphatase Activities in Real-Time.

Cell signaling is governed by dynamic changes in kinase and phosphatase activities, which are difficult to assess with discontinuous readout methods. Here, we introduce an NMR-based reporter approach to directly identify active kinases and phosphatases in complex physiological environments such as cell lysates and to measure their individual activities in a semicontinuous fashion. Multiplexed NMR profiling of reporter phosphorylation states provides unique advantages for kinase inhibitor studies and reveals reversible modulations of cellular enzyme activities under different metabolic conditions.

Probing the recognition of post-translational modifications by combining sortase-mediated ligation and phage-assisted selection.

Reversible post-translational modifications (PTMs) are key regulators of protein function and modulate a multitude of protein-protein interactions in signal transduction networks. Here, we describe a strategy for determining the modification preferences of PTM-binding proteins with only minimal protein amounts that can be obtained by immunoprecipitation from mammalian cell lysates. This method bases on the combination of sortase-mediated ligation and phage-assisted selection strategies.

Facile synthesis of colorimetric histone deacetylase substrates.

Here we report a simple procedure for generating colorimetric histone deacetylase (HDAC) substrates by solid-phase peptide synthesis based on racemization-free couplings of amino acid chlorides. We demonstrate the applicability of these substrates in HDAC assays.

In-cell NMR in mammalian cells: part 1.

Many mammalian IDPs exert important biological functions in key cellular processes and often in highly specialized subsets of cells. For these reasons, tools to characterize the structural and functional characteristics of IDPs inside mammalian cells are of particular interest. Moving from bacterial and amphibian in-cell NMR experiments to mammalian systems offers the unique opportunity to advance our knowledge about general IDP properties in native cellular environments.

Differential contribution of the repeats to heparin binding of HBHA, a major adhesin of Mycobacterium tuberculosis.

Background: Tuberculosis remains one of the most important causes of global mortality and morbidity, and the molecular mechanisms of the pathogenesis are still incompletely understood. Only few virulence factors of the causative agent Mycobacterium tuberculosis are known. One of them is the heparin-binding haemagglutinin (HBHA), an important adhesin for epithelial cells and an extrapulmonary dissemination factor.

NMR profiling of histone deacetylase and acetyl-transferase activities in real time.

Histone deacetylases (HDACs) and histone acetyl-transferases (HATs) are universal regulators of eukaryotic transcriptional activity and emerging therapeutic targets for human diseases. Here we describe the generation of isotope-labeled deacetylation and acetylation reporters for simultaneous NMR readouts of multiple deacetylation and acetylation reactions at different histone H4 sites. The site preferences of two prototypic histone deacetylases (Sir2.

Simultaneous detection of protein phosphorylation and acetylation by high-resolution NMR spectroscopy.

Post-translational protein modifications (PTMs) such as phosphorylation and acetylation regulate a large number of eukaryotic signaling processes. In most instances, it is the combination of different PTMs that "encode" the biological outcome of these covalent amendments in a highly dynamic and cell-state-specific manner. Most research tools fail to detect different PTMs in a single experiment and are unable to directly observe dynamic PTM states in complex environments such as cell extracts or intact cells.