Improving HyperCEST performance by favorable xenon exchange conditions in liposomal nanocarriers.

MRI reporters that combine signal enhancement from saturation transfer with hyperpolarized 129 Xe show nanomolar detection sensitivity for in vitro studies. However, they need further improvement for accelerated CEST build-up that sufficiently dominates the intrinsic loss of hyperpolarization under in vivo conditions. This study introduces liposomes with a HyperCEST-active lipopeptide to enhance the efficiency of a well known Xe host, CrA-ma, with medium Xe exchange kinetics in aqueous environment, by two orders of magnitude. The depolarization time for constant saturation power but increasing saturation time is used as a comparative measure to rank different nanocarrier formulations. A variable cage load illustrates that the available CEST sites should be well distributed throughout the nanocarriers to avoid inefficiency from back exchange. For a liposome loading with only 2 mol% CrA-lipopeptide, the higher exchange kinetics allowed us to work even with 17-fold lower saturation power than for CrA-ma itself to achieve significant image contrast with 129 Xe. Overall, this study illustrates the wide parameter space that is now available when incorporating CrA-labelled lipopeptides into liposomal carriers.

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis.

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome-wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis Relative quantification of the changes in the lysine acetylation levels was determined on a proteome-wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1-like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar-localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss-of-function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low-light conditions.

A miniaturized readout strategy for endogenous histone deacetylase activity.

Histone deacetylases are important drug targets, which are difficult to characterize due to their poor accessibility. We have developed a miniaturized assay for the multi-site readout of deacetylase activity and profiled the substrate selectivity of HDACs for acetylation sites on histone H4 and tumor suppressor protein p53.

Analysis of phosphorylation-dependent protein-protein interactions of histone h3.

Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails.

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.

Site-specific mapping and time-resolved monitoring of lysine methylation by high-resolution NMR spectroscopy.

Methylation and acetylation of protein lysine residues constitute abundant post-translational modifications (PTMs) that regulate a plethora of biological processes. In eukaryotic proteins, lysines are often mono-, di-, or trimethylated, which may signal different biological outcomes. Deconvoluting these different PTM types and PTM states is not easily accomplished with existing analytical tools. Here, we demonstrate the unique ability of NMR spectroscopy to discriminate between lysine acetylation and mono-, di-, or trimethylation in a site-specific and quantitative manner. This enables mapping and monitoring of lysine acetylation and methylation reactions in a nondisruptive and continuous fashion. Time-resolved NMR measurements of different methylation events in complex environments including cell extracts contribute to our understanding of how these PTMs are established in vitro and in vivo.