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. 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.

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. This method can be used to analyze the type of modification that mediates the interaction as well as the influence of the amino acids flanking the modification sites. We have demonstrated the applicability of this method by probing the interaction of phosphorylated tyrosine and serine residues with their respective binding domains.

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. This is never more relevant than for IDPs that exhibit pathological structural rearrangements under certain cellular conditions, as is the case for human α-synuclein in dopaminergic neurons of the substantia nigra in the course of Parkinson's disease, for example. To efficiently deliver isotope-labeled IDPs into mammalian cells is one of the first challenges when preparing a mammalian in-cell NMR sample. The method presented here provides a detailed protocol for the transduction of isotope-labeled α-synuclein, as a model IDP, into cultured human HeLa cells. Cellular IDP delivery is afforded by action of a cell-penetrating peptide (CPP) tag. In the protocol outlined below, the CPP tag is "linked" to the IDP cargo moiety via an oxidative, disulfide-coupling reaction.

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. HBHA mediates mycobacterial adherence to epithelial cells via the interactions of its C-terminal, lysine rich repeat domain with sulfated glycoconjugates on the surface of epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Using defined heparin sulfate (HS) analogs, we determined the minimal heparin fragment length for HBHA binding and structural adaptations of the HBHA heparin-binding domain (HBD) upon binding to heparin. The NMR studies show significant shifts of all residues in the HBD upon interaction with heparin, with stronger shifts in the last repeats compared to the upstream repeats, and indicated that the HS fragments with 14 sugar units cover the entire C-terminal lysine-rich domain of HBHA. The differential implication of the repeats is determined by the relative position of prolines and lysines within each repeat, and may contribute to binding specificity. GAG binding induces a non-homogeneous structural rearrangement in the HBD, with stabilization of a nascent α-helix only in the last penta-repeats. CONCLUSION/SIGNIFICANCE: Mycobacterial HBHA undergoes structural adaptation upon interaction with GAGs, which is likely involved in binding specificities of the adhesin, and mycobacterial pathogens may use HBD polymorphisms for host or organ specificity. Further studies will aim at decoding the complementarity between HBD repeats and HS sequence.

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 (Sir2.1 and HDAC8) and two acetyl-transferases (HAT1 and p300/CBP) were studied in intramolecular competition assays. We identify a previously ill-defined acetylation site, lysine 20 of histone H4, as a preferred target of three of theses enzymes. In situ analyses of endogenous deacetylation reactions at H4 sites in HeLa nuclear extracts point to abundant HDAC activities in human cellular environments.

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. Here we describe in situ observations of phosphorylation and acetylation reactions by high-resolution liquid-state NMR spectroscopy. We delineate the NMR characteristics of progressive lysine acetylation and provide in vitro examples of joint phosphorylation and acetylation events and how they can be deciphered on a residue-specific basis and in a time-resolved and quantitative manner. Finally, we extend our NMR investigations to cellular phosphorylation and acetylation events in human cell extracts and demonstrate the unique ability of NMR spectroscopy to simultaneously report the establishment of these PTMs by endogenous cellular enzymes.