Critical assessment of side-chain conformational space sampling procedures designed for quantifying the effect of side-chain environment

We introduce a family of procedures designed to sample side-chain conformational space at particular locations in protein structures. These procedures (CRSP) use intensive cycles of random assignment of side-chain conformations followed by minimization to determine all the conformations that a group of side-chains can adopt simultaneously. First, we consider a procedure evolving in the dihedral space (dCRSP). Our results suggest that it can accurately map low-energy conformations adopted by clusters of side-chains of a protein. dCRSP is relatively insensitive to various important parameters, and it is sufficiently accurate to capture efficiently the constraint induced by the environment on the conformations a particular side-chain can adopt. Our results show that dCRSP, compared with molecular dynamics (MD), can overcome the problem of the limited set of conformations reached in a reasonable amount of simulations. Next, we introduce procedures (vCRSP) in which valence angles are relaxed, and we assess how efficiently they quantify the conformational entropy of side-chains in the protein native state. For simple peptides, entropies obtained with vCRSP are fully compatible with those obtained with a Monte Carlo procedure. For side-chains in a protein environment, however, vCRSP appears of limited use. Finally, we consider a two-step procedure that combines dCRSP and vCRSP. Our tests suggest that it is able to overcome the limitations of vCRSP. We also note that dCRSP provides a reasonable initial approximation. This family of procedures offers promise in quantifying the contribution of conformational entropy to the energetics of protein structures.     

Limited proteolysis of human histone deacetylase 1

Background  

Histone deacetylase (HDAC) proteins are associated with cell proliferation, differentiation, apoptosis, and cancer. Specifically, HDAC1 is linked with cell growth, a hallmark of cancer formation. HDAC1 is a phosphoprotein and phosphorylation at S421 and S423 promotes HDAC1 enzymatic activity and protein association. While single and double point mutants of HDAC1 at S421 and S423 appear functionally similar, the evidence suggests that HDAC1 is phosphorylated simultaneously at both S421 and S423 in vivo. Additional experiments are necessary to probe the role of double phosphorylation of HDAC1 at S421 and S423.     

A method for conformational sampling of loops in proteins based on adiabatic decoupling and temperature or force scaling

A method for conformational Boltzmann sampling of loops in proteins in aqueous solution is presented that is based on adiabatic decoupling molecular dynamics (MD) simulation with temperature or force scaling. To illustrate the enhanced sampling, the loop from residues 33 to 43 in the bovine protein ribonuclease A is adiabatically decoupled from the rest of the protein and the solvent with a mass scaling factor s(m) =1000 and the sampling is enhanced with a scaling of the temperature using s(T) =2 or of the force using s(V) =0.667. Over 5 ns of simulation the secondary structure of the protein remains unaltered while a combined dihedral-angle conformational cluster analysis shows an increase of conformations outside the first most populated cluster of loop conformations for adiabatic decoupling MD with temperature scaling using s(T) =2 or force scaling using s(V) =0.667 compared to the standard MD simulation. The atom-positional root-mean-square fluctuations of the C(α) atoms of the loop show an increase in the movement of the loop as well, indicating that adiabatic decoupling MD with upscaling of the temperature or downscaling of the force is a promising method for conformational Boltzmann sampling.     

Proteomic profiling of human colon cancer cells treated with the histone deacetylase inhibitor belinostat

The anticancer drug belinostat is a hydroxamate histone deacetylase inhibitor that has shown significant antitumour activity in various tumour models and also in clinical trials. In this study, we utilized a proteomic approach in order to evaluate the effect of this drug on protein expression in the human colon cancer cell line HCT116. Protein extracts from untreated HCT116 cells, and cells grown for 24 h in the presence of 1 and 10 μM belinostat were analysed by 2‐D gel electrophoresis. Proteins were visualized by colloidal Coomassie blue staining and quantitative analysis of gel images revealed 45 unique differentially expressed proteins that were identified by LC‐MSMS analysis. Among these proteins, of particular interest are the downregulated proteins nucleophosmin and stratifin, and the upregulated proteins nucleolin, gelsolin, heterogeneous nuclear ribonucleoprotein K, annexin 1, and HSP90B that all were related to the proto‐oncogene proteins p53, Myc, activator protein 1, and c‐fos protein. The modulation of these proteins is consistent with the observations that belinostat is able to inhibit clonogenic cell growth of HCT116 cells and the biological role of these proteins will be discussed.     

Aromatic polyketide production in Cordyceps indigotica, an entomopathogenic fungus, induced by exposure to a histone deacetylase inhibitor

Cultivation of Cordyceps indigotica, an entomopathogenic fungus, in the presence of suberoyl bis-hydroxamic acid (an HDAC inhibitor) greatly activated its polyketide synthesis apparatus to afford six novel aromatic polyketides, indigotides C-F (1-4), 13-hydroxyindigotide A (5), and 8-O-methylindigotide B (6). The structures of these compounds were determined by NMR spectroscopic analyses. Among the compounds, indigotides C-E (1-3) possessed unprecedented dimeric polyketide frameworks possibly generated via a [4 + 2] cycloaddition or Michael type reaction.     

Geometric algorithms for the conformational analysis of long protein loops

The efficient filtering of unfeasible conformations would considerably benefit the exploration of the conformational space when searching for minimum energy structures or during molecular simulation. The most important conditions for filtering are the maintenance of molecular chain integrity and the avoidance of steric clashes. These conditions can be seen as geometric constraints on a molecular model. In this article, we discuss how techniques issued from recent research in robotics can be applied to this filtering. Two complementary techniques are presented: one for conformational sampling and another for computing conformational changes satisfying such geometric constraints. The main interest of the proposed techniques is their application to the structural analysis of long protein loops. First experimental results demonstrate the efficacy of the approach for studying the mobility of loop 7 in amylosucrase from Neisseria polysaccharea. The supposed motions of this 17-residue loop would play an important role in the activity of this enzyme.     

Antitumor effect of liposomal histone deacetylase inhibitor-lipid conjugates in vitro

Histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), approved by the Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma, is a promising new treatment strategy for various cancers. In this study, we hypothesized that a liposomal formulation of HDACI might efficiently deliver HDACI into tumors. To incorporate HDACI efficiently into the liposomal membrane, we synthesized six HDACI-lipid conjugates, in which polyethylene glycol(2000) (PEG(2000))-lipid or cholesterol (Chol) was linked with a potent hydroxamic acid, HDACI, SAHA or K-182, by cleavable linkers, such as ester, carbamide and disulfide bonds. Liposomal HDACI-lipid conjugates were prepared with distearoylphosphatidylcholine (DSPC) and HDACI-Chol conjugate or with DSPC, Chol and HDACI-PEG-lipid conjugates, and their cytotoxicities were evaluated for human cervix tumor HeLa and mouse colon tumor Colon 26 cells. Among the liposomes, liposomal oleyl-PEG(2000)-SAHA conjugated with SAHA and oleyl-PEG(2000) via a carbamate linker showed higher cytotoxicity via hyperacetylation of histone H3 and induction of caspase 3/7 activity. These results suggested that liposomal HDACI-lipid conjugates may be a potential tool for cancer therapy.     

Role of histone deacetylase activity in the developing lateral line neuromast of zebrafish larvae

Histone deacetylases are involved in many biological processes and have roles in regulating cell behaviors such as cell cycle entry, cell proliferation and apoptosis. However, the effect of histone deacetylases on the development of hair cells (HCs) has not been fully elucidated. In this study, we examined the influence of histone deacetylases on the early development of neuromasts in the lateral line of zebrafish. Hair cell development was evaluated by fluorescent immunostaining in the absence or presence of histone deacetylase inhibitors. Our results suggested that pharmacological inhibition of histone deacetylases with inhibitors, including trichostatin A, valproic acid and MS-275, reduced the numbers of both HCs and supporting cells in neuromasts. We also found that the treatment of zebrafish larvae with inhibitors caused accumulation of histone acetylation and suppressed proliferation of neuromast cells. Real-time PCR results showed that the expression of both p21 and p27 mRNA was increased following trichostatin A treatment and the increase in p53 mRNA was modest under the same conditions. However, the expression of p53 mRNA was significantly increased by treatment with a high concentration of trichostatin A. A high concentration of trichostatin A also led to increased cell death in neuromasts as detected in a TUNEL assay. Moreover, the nuclei of most of these pyknotic cells were immunohistochemically positive for cleaved caspase-3. These results suggest that histone deacetylase activity is involved in lateral line development in the zebrafish and might have a role in neuromast formation by altering cell proliferation through the expression of cell cycle regulatory proteins.     

Correlation of the side-chain hubs with the functional residues in DNA binding protein structures

The structure networks of DNA-binding proteins have been constructed and analyzed. The detailed analysis of the networks indicates a strong relation between the positions of the residues interacting with DNA and those that form extensive interactions within the protein structure (called hubs). This study shows that the functional residues in these proteins are held in place by efficient scaffolding of the structure using side-chain interactions, thus highlighting the role of these side-chain hubs with respect to the functional residues in the protein structure.     

Multiple scaling replica exchange for the conformational sampling of biomolecules in explicit water

A multiple scaling replica exchange method for the efficient conformational sampling of biomolecular systems in explicit solvent is presented. The method is a combination of the replica exchange with solute tempering (REST) technique and a Tsallis biasing potential. The Tsallis biasing increases the sampling efficiency, while the REST minimizes the number of replicas needed. Unbiased statistics can be obtained by reweighting of the data using a weighted histogram analysis technique. The method is illustrated by its application to a ten residue peptide in explicit water.     

Recent developments in peptide ligation independent of amino acid side-chain functional group

Chemical synthesis of peptides and proteins has evolved into an indispensable tool for chemical biology. Peptide ligation is a straightforward technique for joining two short peptide fragments together via a native peptide bond to afford a larger natural peptide or protein. However, the junction sites are limited to several specific amino acids because most peptide ligations involve participation of the side-chain functional groups of the junction-site amino acids. To overcome such intrinsic limitations, “general” peptide ligations which do not rely on the side-chain functional group have been developed. This review summarized the recent developments in peptide ligations that are independent of side-chain functional group of ligation-junction-site amino acid.     

Macrolactamization versus macrolactonization: total synthesis of FK228, the depsipeptide histone deacetylase inhibitor

The cyclic depsipeptide FK228 is the only natural product histone deacetylase (HDAC) inhibitor that has advanced to clinical trials as an anticancer agent. While currently obtained by fermentation, total synthesis is an attractive alternative that will facilitate the preparation of unnatural analogues. The previous total syntheses of FK228 featured macrocylization by ester bond formation from a seco-hydroxy acid. Such routes are operationally jeopardized by the steric hindrance of the carboxylic acid and the sensitivity of the allylic alcohol toward elimination. We report a strategically different approach whereby the ester bond is formed intermolecularly at an early stage and macrocyclization is efficiently achieved by amide bond formation.     

Improvements to the distance geometry algorithm for conformational sampling of cyclic structures

Modifications to the distance geometry algorithm as embodied in the program DGEOM have been made to improve sampling capabilities. Specifically, torsion angle sampling replaces distance sampling for 1,4 atomic relationships and correlated distance sampling is disabled. The effects of these modifications are illustrated by comparing the different sets of conformations produced for butane. In addition, these changes are shown to increase the conformational sampling of two medium-sized rings, cycloheptadecane and caprylolactam. The current results for these molecules are compared to those of other conformational searching methods.     

Psammaplins from the sponge Pseudoceratina purpurea: inhibition of both histone deacetylase and DNA methyltransferase

Four novel bisulfide bromotyrosine derivatives, psammaplins E (9), F (10), G (11), and H (12), and two new bromotyrosine derivatives, psammaplins I (13) and J (14), were isolated from the sponge Pseudoceratina purpurea, along with known psammaplins A (4), B (6), C (7), and D (8) and bisaprasin (5). The structures of psammaplins E (9) and F (10), which each contain an oxalyl group rarely found in marine organisms, were determined by spectroscopic analysis. Compounds 4, 5, and 10 are potent histone deacetylase inhibitors and also show mild cytotoxicity. Furthermore, compounds 4, 5, and 11 are potent DNA methyltransferase inhibitors. The biogenetic pathway previously proposed for the psammaplins class is also revisited.     

Effects of external electromagnetic fields on the conformational sampling of a short alanine peptide

Non‐equilibrium molecular dynamics simulations of a solvated 21‐residue polyalanine (A21) peptide, featuring a high propensity for helix formation, have been performed at 300 K and 1 bar in the presence of external electromagnetic (e/m) fields in the microwave region (2.45 GHz) and an r.m.s. electric field intensity range of 0.01–0.05 V/Å. To investigate how the field presence affects transitions between the conformational states of a protein, we report 16 independent 40 ns‐trajectories of A21 starting from both extended and fully folded states. We observe folding‐behavior of the peptide consistent with prior simulation and experimental studies. The peptide displays a natural tendency to form stable elements of secondary structure which are stabilized by tertiary interactions with proximate regions of the peptide. Consistent with our earlier work, the presence of external e/m fields disrupts this behavior, involving a mechanism of localized dipolar alignment which serves to enhance intra‐protein perturbations in hydrogen bonds (English, et al., J. Chem. Phys. 2010 , 133, 091105), leading to more frequent transitions between shorter‐lifetime states. © 2012 Wiley Periodicals, Inc.