Marek Cebrat

Structure of the GCN5 histone acetyltransferase bound to a bisubstrate inhibitor

Histone acetyltransferases (HATs) use acetyl CoA to acetylate target lysine residues within histones and other transcription factors, such as the p53 tumor suppressor, to promote gene activation. HAT enzymes fall into subfamilies with divergence in sequence and substrate preference. Several HAT proteins have been implicated in human cancer. We have previously reported on the preparation of peptide-CoA conjugate inhibitors with distinct specificities for the p300/CBP [cAMP response element binding protein (CREB)-binding protein] or GCN5 HAT subfamilies. Here we report on the crystal structure of the GCN5 HAT bound to a peptide-CoA conjugate containing CoA covalently attached through an isopropionyl linker to Lys-14 of a 20-aa N-terminal fragment of histone H3. Surprisingly, the structure reveals that the H3 portion of the inhibitor is bound outside of the binding site for the histone substrate and that only five of the 20 aa residues of the inhibitor are ordered. Rearrangements within the C-terminal region of the GCN5 protein appear to mediate this peptide displacement. Mutational and enzymatic data support the hypothesis that the observed structure corresponds to a late catalytic intermediate. The structure also provides a structural scaffold for the design of HAT-specific inhibitors that may have therapeutic applications for the treatment of HAT-mediated cancers.

Histone Acetyltransferase Activity of p300 Is Required for Transcriptional Repression by the Promyelocytic Leukemia Zinc Finger Protein

ABSTRACT Histone acetyltransferase (HAT) activities of proteins such as p300, CBP, and P/CAF play important roles in activation of gene expression. We now show that the HAT activity of p300 can also be required for down-regulation of transcription by a DNA binding repressor protein. Promyelocytic leukemia zinc finger (PLZF), originally identified as a fusion with retinoic acid receptor alpha in rare cases of all-trans-retinoic acid-resistant acute promyelocytic leukemia, is a transcriptional repressor that recruits histone deacetylase-containing corepressor complexes to specific DNA binding sites. PLZF associates with p300 in vivo, and its ability to repress transcription is specifically dependent on HAT activity of p300 and acetylation of lysines in its C-terminal C2-H2 zinc finger motif. An acetylation site mutant of PLZF does not repress transcription and is functionally deficient in a colony suppression assay despite retaining its abilities to interact with corepressor/histone deacetylase complexes. This is due to the fact that acetylation of PLZF activates its ability to bind specific DNA sequences both in vitro and in vivo. Taken together, our results indicate that a histone deacetylase-dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a coactivator protein in transcriptional repression.

Cyclolinopeptide A (CLA) mediates its immunosuppressive activity through cyclophilin-dependent calcineurin inactivation

The immunosuppressive cyclic nonapeptide cyclolinopeptide A inhibits calcium‐dependent, but not calcium‐independent, activation of T lymphocytes comparably to the actions of cyclosporin A and FK506. The concentration required for complete inhibition, however, is 10 times higher than that of cyclosporin A. In addition, we demonstrate that calcineurin, a phosphatase which plays an important role in T lymphocyte signalling, is inhibited in vitro by cyclolinopeptide A by a mechanism dependent on the peptidyl‐prolyl cis‐trans isomerase (PPIase) cyclophilin A but not FKBP12. Direct binding of cyclolinopeptide A to cyclophilin A was confirmed using tryptophan fluorescence studies and PPIase assays. These results represent a third example of the production of a natural product that neutralises calcineurin by a mechanism dependent on the primary binding to a PPIase.

Selective HAT Inhibitors as Mechanistic Tools for Protein Acetylation

Publisher Summary This chapter introduces the use of selective Histone acetyltransferases (HATs) inhibitors as mechanistic tools to probe the catalytic features of HATs and their roles in various cellular pathways. The chapter describes the design, synthesis, and applications of these inhibitors in different biological contexts. HATs regulate gene expression by the targeted acetylation of histones and other proteins. It is a difficult challenge to identify the functional effects of protein acetylation in specific pathways. HATs catalyze the transfer of acetyl groups from acetyl-CoA to the ɛ-amino groups of lysine residues in protein and play a major role in the regulation of transcriptional machinery and the modulation of gene expression. While HATs were initially thought of as enzymes that catalyze the acetylation of histones on lysine residues, it is increasingly recognized that these inhibitors act on a wide variety of protein substrates. These acetylation events may in some cases affect protein-protein interactions. The use of small molecules to modulate cellular and in vivo systems, sometimes called ‘‘chemical genetics,’’ has received renewed interest in the biology community in part because of continuing improvements in synthetic chemistry as well as molecular design.

The immunosuppressive activity and solution structures of ubiquitin fragments

Recently, ubiquitin was suggested as a promising anti-inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well-defined conformation in methanol, its structure was distinct from the corresponding 50-59 fragment in the native ubiquitin molecule. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 423-431, 2009.

The problem of amino acid complementarity and antisense peptides.

The review presents three hypotheses concerning the amino acid complementarity: 1) the Mekler-Blalock antisense hypothesis; 2) the Root-Bernstein approach based on stereochemical complementarity of amino acids and anti-amino acids coded by anticodons read in parallel with the coding DNA strand; 3) Siemion hypothesis resulting from the periodicity of the genetic code. The current state of knowledge as well as the results of the implementations of these hypotheses are compared. A special attention is given to Root-Bernstein and Siemion hypotheses, which differ in only few points of the complementarity prediction. We describe methods of investigation of peptide-antipeptide pairing, including circular dichroism, mass spectrometry, affinity chromatography and other techniques. The biological applications of complementarity principle are considered, such as search for bioeffector-bioreceptor interaction systems, the influence of peptide-antipeptide pairing on the activity of peptide hormones, and the application of antipeptides in immunochemistry. The possible role of amino acid-anti-amino acid interactions in the formation of the spatial structures of peptides, proteins and protein complexes is discussed. Such problems as the pairing preferences of protein-protein interfaces, the role of the pairing in the creation of disulfide bonds and the possible appearance of such interactions in beta-structure are also examined. The main intention of the paper is to bring the complementarity problem to the attention of the scientific community, as a possible tool in proteomics, molecular design and molecular recognition.

Immunosuppressive activity of hymenistatin I.

Hymenistatin I (HS-I), a cyclic octapeptide [c-(-Pro-Pro-Tyr-Val-Pro-Leu-Ile-Ile-)], was synthesized by the solid-phase peptide synthesis method and examined for its immunosuppressive activity in the humoral and cellular immune responses. The peptide activity was tested on cell lines producing various cytokines. The results are compared with the activity of the well-known immunosuppressive agent cyclosporin A (CsA). It was found that hymenistatin I exerts immunosuppressive effect (both in the humoral and cellular immune responses) comparable with that of CsA. Comparison of the influence of HS-I and CsA on cytokines production suggests that the mechanisms of the interaction with the immunological system are substantially different for the two compounds tested.

The peptide molecular links between the central nervous and the immune systems.

Summary.The central nervous system (CNS) and the immune system were for many years considered as two autonomous systems. Now, the reciprocal connections between them are generally recognized and very well documented. The links are realized mainly by various immuno- and neuropeptides. In the review the influence of the following immunopeptides on CNS is presented: tuftsin, thymulin, thymopoietin and thymopentin, thymosins, and thymic humoral factor. On the other side, the activity in the immune system of such neuropeptides as substance P, neurotensin, some neurokinins, enkephalins, and endorphins is discussed.

Peptides derivatized with bicyclic quaternary ammonium ionization tags. Sequencing via tandem mass spectrometry

Improving the sensitivity of detection and fragmentation of peptides to provide reliable sequencing of peptides is an important goal of mass spectrometric analysis. Peptides derivatized by bicyclic quaternary ammonium ionization tags: 1-azabicyclo[2.2.2]octane (ABCO) or 1,4-diazabicyclo[2.2.2]octane (DABCO), are characterized by an increased detection sensitivity in electrospray ionization mass spectrometry (ESI-MS) and longer retention times on the reverse-phase (RP) chromatography columns. The improvement of the detection limit was observed even for peptides dissolved in 10 mM NaCl. Collision-induced dissociation tandem mass spectrometry of quaternary ammonium salts derivatives of peptides showed dominant a- and b-type ions, allowing facile sequencing of peptides. The bicyclic ionization tags are stable in collision-induced dissociation experiments, and the resulted fragmentation pattern is not significantly influenced by either acidic or basic amino acid residues in the peptide sequence. Obtained results indicate the general usefulness of the bicyclic quaternary ammonium ionization tags for ESI-MS/MS sequencing of peptides.

Hydrogen-deuterium exchange in imidazole as a tool for studying histidine phosphorylation

Isotope exchange at the histidine C2 atom of imidazole in D2O solution is well known to occur at a significantly slower rate than the exchange of amide protons. Analysis of the kinetics of this isotope-exchange reaction is proposed herein as a method of detecting histidine phosphorylation. This modification of His-containing peptides is challenging to pinpoint because of its instability under acidic conditions as well as during CID-MS analysis. In this work, we investigated the effect of phosphorylation of the histidine side chain in peptides on deuterium–hydrogen exchange (DHX) in the imidazole. The results demonstrate that phosphorylation dramatically slows the rate of the DHX reaction. This phenomenon can be applied to detect phosphorylation of peptides at the histidine residue (e.g., in enzymatic digests). We also found that the influence of the peptide sequence on the exchange kinetics is relatively small. A CID fragmentation experiment revealed that there was no detectable hydrogen scrambling in peptides deuterated at C2 of the imidazole ring. Therefore, MS/MS can be used to directly identify the locations of deuterium ions incorporated into peptides containing multiple histidine moieties.