Lysine Deacetylase Substrate Selectivity: A Dynamic Ionic Interaction Specific to KDAC8.

Lysine acetylation and deacetylation are critical for regulation of many cellular proteins. Despite the importance of this cycle, it is unclear how lysine deacetylase (KDAC) family members discriminate between acetylated proteins to react with a discrete set of substrates. Potential short-range interactions between KDAC8 and a known biologically relevant peptide substrate were identified using molecular dynamics (MD) simulations. Activity assays with a panel of peptides derived from this substrate supported a putative ionic interaction between arginine at the -1 substrate position and KDAC8 D101. Additional assays and MD simulations confirmed this novel interaction, which promotes deacetylation of substrates. Verification that a negatively charged residue at the 101 position is necessary for the ionic interaction and observed reactivity with the substrates was performed using KDAC8 derivatives. Notably, this interaction is specific to KDAC8, as KDAC1 and KDAC6 do not form this interaction and each KDAC has a different specificity profile with the peptide substrates, even though all KDACs could potentially form ionic interactions. When reacted with a panel of putative human KDAC substrates, KDAC8 preferentially deacetylated substrates containing an arginine at the -1 position. KDAC8 D101-R(-1) is a specific enzyme-substrate interaction that begins to explain how KDACs discriminate between potential substrates and how different KDAC family members can react with different subsets of acetylated proteins in cells. This multi-pronged approach will be extended to identify other critical interactions for KDAC8 substrate binding and determine critical interactions for other KDACs.

Cloning, purification, and homology modeling of Histone deacetylase in Leishmania donovani.

Neglected diseases, such as leishmaniasis, are still a major health problem in poor countries. To date, there is a severe lack of effective, safe, and affordable treatment for leishmaniasis. Currently, there are very limited chemotherapeutic options, and the development of vaccines is still underway. Hence, novel therapeutic strategies need to be developed against leishmanial parasites. Histone deacetylases (HDACs), silent regulators of many critical pathways, have been validated as potential therapeutic targets in cancer and several parasitic diseases. In the present work, we have isolated and characterized biologically active Zn2+-dependent HDAC protein from leishmania that can be studied further as a potential anti-leishmanial drug target to develop new therapies against neglected diseases. The nucleotide sequence of the HDAC gene with no intervening sequence was amplified, cloned in a pET-28a vector, and later transformed into the BL21(DE3) competent E. coli bacterial cells. After transformation, the cells were cultured and induced with 0.6 mM of IPTG to express histidine-tagged HDAC protein (LD_HDAC), which was later purified using nickel affinity chromatography. The approximate protein size confirmed with the help of 10% SDS-PAGE was ~48.0 kDa. The enzymatic assay using the purified protein confirmed it as biologically active. A three dimensional structure of LD_HDAC was modeled using the crystal structure of HDAC2 protein of Homo sapiens (PDB ID: 6G3O). This protein can be utilized for the screening of Leishmania-specific HDAC inhibitors.

Purification of metal-dependent lysine deacetylases with consistently high activity.

Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation. Characterization of KDAC activity and substrate identification is complicated by inconsistent activity of prepared enzyme and a range of multi-step purifications. We describe a simplified protocol based on two-step affinity chromatography. The purification method is appropriate for use regardless of expression host, and we demonstrate purification of several representative members of the KDAC family as well as a selection of mutated variants. The purified proteins are highly active and consistent across preparations.

Expression, purification and characterization of the human MTA2-RBBP7 complex.

The repressive Nucleosome Remodeling and histone Deacetylation (NuRD) complex remodels the chromatin structure by coupling ATP-dependent remodeling activity with histone deacetylase function and plays important roles in regulating gene transcription, DNA damage repair and chromatin assembly. The complex is composed of six subunits: Metastasis Associated proteins MTA1/2/3 initially recruit histone chaperones RBBP4/7 followed by the histone deacetylases HDAC1/2 forming a core complex. Further association of the CpG-binding protein MBD2/3, p66α/ß and the ATP-dependent helicase CDH3/4 constitutes the NuRD complex. Recent structural studies on truncated human proteins or orthologous have revealed that the stoichiometry of the MTA1-RBBP4 complex is 2:4. This study reports expression and purification of the intact human MTA2-RBBP7 complex using HEK293F cells as expression system. In analogy with findings on the Drosophila NuRD complex, we find that also the human MTA-RBBP can be isolated in vitro. Taken together with previous findings this suggests, that MTA-RBBP is a stable complex, with a central role in the initial assembly of the human NuRD complex. Refined 3D volumes of the complex generated from negative stain electron microscopy (EM) data reveals an elongated architecture that is capable of hinge like motion around the center of the particle.

Histone deacetylase activity assay.

Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from the ε-amino groups of conserved lysine residues in the amino terminal tail of histones. Accumulating evidence suggests that many, if not all, HDACs can also deacetylate nonhistone proteins. Through deacetylating histones and nonhistone proteins, HDACs regulate a variety of cellular processes including gene transcription, cell differentiation, DNA damage responses, and apoptosis. Aberrant HDACs are implicated in many human diseases and, therefore, it is important to have a consistent and reliable assay for analyzing HDAC activities. The focus of this chapter is to provide up-to-date, easy-to-follow, approaches and techniques, for the assay of HDAC enzymatic activities.

Aurora B-dependent regulation of class IIa histone deacetylases by mitotic nuclear localization signal phosphorylation.

Class IIa histone deacetylases (HDACs 4/5/7/9) are transcriptional regulators with critical roles in cardiac disease and cancer. HDAC inhibitors are promising anticancer agents, and although they are known to disrupt mitotic progression, the underlying mechanisms of mitotic regulation by HDACs are not fully understood. Here we provide the first identification of histone deacetylases as substrates of Aurora B kinase (AurB). Our study identifies class IIa HDACs as a novel family of AurB targets and provides the first evidence that HDACs are temporally and spatially regulated by phosphorylation during the cell cycle. We define the precise site of AurB-mediated phosphorylation as a conserved serine within the nuclear localization signals of HDAC4, HDAC5, and HDAC9 at Ser265, Ser278, and Ser242, respectively. We establish that AurB interacts with these HDACs in vivo, and that this association increases upon disruption of 14-3-3 binding. We observe colocalization of endogenous, phosphorylated HDACs with AurB at the mitotic midzone in late anaphase and the midbody during cytokinesis, complemented by a reduction in HDAC interactions with components of the nuclear corepressor complex. We propose that AurB-dependent phosphorylation of HDACs induces sequestration within a phosphorylation gradient at the midzone, maintaining separation from re-forming nuclei and contributing to transcriptional control.

Arabidopsis thaliana histone deacetylase 14 (HDA14) is an α-tubulin deacetylase that associates with PP2A and enriches in the microtubule fraction with the putative histone acetyltransferase ELP3.

It is now emerging that many proteins are regulated by a variety of covalent modifications. Using microcystin-affinity chromatography we have purified multiple protein phosphatases and their associated proteins from Arabidopsis thaliana. One major protein purified was the histone deacetylase HDA14. We demonstrate that HDA14 can deacetylate α-tubulin, associates with α/ß-tubulin and is retained on GTP/taxol-stabilized microtubules, at least in part, by direct association with the PP2A-A2 subunit. Like HDA14, the putative histone acetyltransferase ELP3 was purified on microcystin-Sepharose and is also enriched at microtubules, potentially functioning in opposition to HDA14 as the α-tubulin acetylating enzyme. Consistent with the likelihood of it having many substrates throughout the cell, we demonstrate that HDA14, ELP3 and the PP2A A-subunits A1, A2 and A3 all reside in both the nucleus and cytosol of the cell. The association of a histone deacetylase with PP2A suggests a direct link between protein phosphorylation and acetylation.

Expression, purification, and S-nitrosylation of recombinant histone deacetylase 8 in Escherichia coli.

Histone deacetylase (HDAC) 8 is a zinc ion dependent enzyme involved in removing the acetyl group from the core histones and other proteins which belong to Class I HDACs. It was reported that nitric oxide (NO) is a key regulator of HDAC function and S-nitrosylation of HDAC2 induces chromatin remodelling in neurons. This work reports the successful recombinant expression of human HDAC8 in Escherichia coli with two plasmids and the purification and S-nitrosylation in vitro. It was found that HDAC8 can be S-nitrosylated by the NO donor S-nitrosoglutathione (GSNO) in vitro, and the activity of HDAC8 was significantly inhibited when incubated with GSNO and S-nitrosocysteine in a time- and dosage-dependent manner, but sodium nitroprusside (SNP), and dithiothreitol cannot reverse this inhibition. These observations support and extend the concept that NO may regulate HDAC8 function by S-nitrosylation.

Evidence for the phosphorylation of serine259 of histone deacetylase 5 by protein kinase Cδ.

Signaling via pro-growth G protein coupled receptors triggers phosphorylation of HDAC5 on two serine residues (Ser259 and Ser498), resulting in nuclear export of HDAC5 and de-repression of downstream target genes. In the previous paper we reported the important role of PKD isozymes in the regulation of HDAC5 by phosphorylating Ser498 of HDAC5 [Q.K. Huynh, T.A. Mckinsey, Arch. Biochem. Biophys. 450 (2006) 141-148]. In the present paper, we provide evidence that PKCδ can directly phosphorylate Ser259 of HDAC5. The evidence is based on the following facts (a) isolated kinase fraction from human failing heart tissues contained PKCδ that phosphorylated HDAC5 Ser259 peptide and no significant activity was found for the unbound fraction after they were immunoprecipitated with PKCδ specific antibody; (b) specific inhibitors for PKCδ inhibited kinase activity from isolated fraction and recombinant human PKCδ with similar IC50 values; (c) recombinant human PKCδ can directly phosphorylate full length Ser259 HDAC5 protein and HDAC5 Ser259 peptide. The results suggest that in addition to activation of protein kinase D isozymes by phosphorylating Ser744 and Ser748 at their activation sites, PKCδ may also play a role in the regulation of HDAC5 by phosphorylation of Ser259.

Detecting protein-small molecule interactions using fluorous small-molecule microarrays.

General binding assays involving microarrays of small molecules can be used to identify small molecule ligands for nearly any protein, even in the absence of knowledge about protein structure or function. Several suitable methods for manufacturing small molecule microarrays (SMMs) exist and different immobilization methods may be more or less preferable for any given application. Here, we describe a protocol for noncovalent and homogenous capture of small molecules using fluorous interactions between small molecules containing fluorocarbon tags and fluorocarbon-coated glass surfaces. These arrays are especially useful for applications that require display of compounds in a specific orientation such as screening biased libraries.

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 muM 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.

Histone deacetylase activity assay.

Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from the epsilon-amino groups of conserved lysine residues in the amino terminal tail of histones. In humans, there are 18 potential deacetylase enzymes that are responsible for the removal of acetyl groups and maintenance of the equilibrium of lysine acetylation on histones. Like most histone modification enzymes, accumulating evidence suggests that many, if not all, HDACs can also modify non-histone proteins. The focus of this article is to provide up-to-date, easy to follow, approaches and techniques specifically for the assay of HDAC enzymatic activities.

Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitro.

At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.

Plasmodium falciparum Sir2: an unusual sirtuin with dual histone deacetylase and ADP-ribosyltransferase activity.

In the human malaria parasite Plasmodium falciparum, a member of the sirtuin family has been implicated in the epigenetic regulation of virulence genes that are vital to malaria pathogenesis and persistence. This eukaryotic sirtuin, PfSir2, is divergent in sequence from those characterized thus far and belongs to the phylogenetic class that contains primarily eubacterial and archaeal sirtuins. PfSir2 cofractionates with histones in blood-stage parasites, and the recombinant enzyme efficiently deacetylates the N-terminal tails of histones H3 and H4. In addition, PfSir2 can ADP-ribosylate both histones and itself, an activity that is minimal or absent in most sirtuins with significant deacetylase activity. Strikingly, the deacetylase activity of PfSir2 is dependent on its ADP-ribosylation. Finally, although PfSir2 is not affected by established sirtuin inhibitors, it can be completely inhibited by nicotinamide, a natural product of the sirtuin reaction. This study shows that PfSir2 has the appropriate characteristics to be a direct regulator of chromatin structure in P. falciparum. It also raises the significant possibility that both ADP-ribosylation and deacetylation of histones could be sirtuin-regulated modulators of chromatin structure in this species.

Enhancement of cisplatin induced apoptosis by suberoylanilide hydroxamic acid in human oral squamous cell carcinoma cell lines.

Both the resistance of tumor cells to cisplatin and dose-related toxicity remain two of the most important problems in the chemotherapy of clinical oral squamous cell carcinoma (OSCC). Researchers have been seeking a combinative treatment regimen to improve the effect of chemotherapy. As potent new anti-cancer drugs, histone deacetylase inhibitors (HDACI(S)) have been reported to be associated with chromatin modification and display synergistic activities with some traditional chemotherapeutic agents. In this study, we evaluated the potential combinative effect of low dose cisplatin and suberoylanilide hydroxamic acid (SAHA, one of the most potent HDACI(S)) in OSCC cell lines. Cell viability and apoptotic assay were examined. Compared with either cisplatin (4 microg/ml) or SAHA (2 microM) treated alone, co-administration of both drugs synergistically induces cytotoxicity and apoptosis in both Tca8113 and KB cell lines. Furthermore, diverse apoptosis-associated proteins, including p53, BID, cytochrome C and caspase-3 were involved in the induction of apoptosis. Our results suggest that concurrent treatment with SAHA enhances tumor cell sensitivity to subtoxic doses of cisplatin. This may be regarded as a novel strategy for treatment of OSCC.

One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system.

Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M(r):1000-8000) and dextran (M(r):40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.

DNMT1 and HDAC1 gene expression in impaired spermatogenesis and testicular cancer.

DNA methylation catalyzed by DNA methyltransferases (DNMTs) and histone deacetylation catalyzed by histone deacetylases (HDACs) play an important role for the regulation of gene expression during carcinogenesis and spermatogenesis. We therefore studied the cell-specific expression of DNMT1 and HDAC1 for the first time in human testicular cancer and impaired human spermatogenesis. During normal spermatogenesis, DNMT1 and HDAC1 were colocalized in nuclei of spermatogonia. While HDAC1 was additionally present in nuclei of Sertoli cells, DNMT1 was restricted to germ cells exhibiting a different expression pattern of mRNA (in pachytene spermatocytes and round spermatids) and protein (in round spermatids). Interestingly, in infertile patients revealing round spermatid maturation arrest, round spermatids lack DNMT1 protein, while pachytene spermatocytes became immunopositive for DNMT1. In contrast, no changes in the expression pattern could be observed for HDAC1. This holds true also in testicular tumors, where HDAC1 has been demonstrated in embryonal carcinoma, seminoma and teratoma. Interestingly, DNMT1 was not expressed in seminoma, but upregulated in embryonal carcinoma.

Preparation of enzymatically active recombinant class III protein deacetylases.

Class III histone deacetylases, or sirtuins, are homologous to the Saccharomyces cerevisiae transcriptional regulator SIR2. The class III enzymes are characterized by their dependence on nicotinamide adenine dinucleotide (NAD+). This cofactor serves as an acetyl-group acceptor in the deacetylation reaction generating O-acetyl-ADP-ribose. Enzymatic activity of sirtuin can be measured in vitro using recombinant proteins purified from mammalian cells after overexpression or after purification from Escherichia coli. This review discusses protocols for the purification of enzymatically active human sirtuin 1, 2, and 3 and their activities on histone and nonhistone substrates.