Inhibitory effects of extracts from Eucalyptus gunnii on α-synuclein amyloid fibrils.

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Despite the numerous studies on the inhibition of amyloid formation, the prevention and treatment of a majority of amyloid-related disorders are still challenging. In this study, we investigated the effects of various plant extracts on amyloid formation of α-synuclein. We found that the extracts from Eucalyptus gunnii are able to inhibit amyloid formation, and to disaggregate preformed fibrils, in vitro. The extract itself did not lead cell damage. In the extract, miquelianin, which is a glycosylated form of quercetin and has been detected in the plasma and the brain, was identified and assessed to have a moderate inhibitory activity, compared to the effects of ellagic acid and quercetin, which are strong inhibitors for amyloid formation. The properties of miquelianin provide insights into the mechanisms controlling the assembly of α-synuclein in the brain.

Total Synthesis and Structural Characterization of Caveolin-1.

Caveolin-1, which is an essential protein for caveola formation, was chemically synthesized. It is composed of 177 amino acid residues, is triply palmitoylated at the C-terminal region, and is inserted into the lipid bilayer to form a V-shaped structure in the middle of the polypeptide chain. The entire sequence was divided into five peptide segments, each of which was synthesized by the solid-phase method. To improve the solubility of the C-terminal region, O-acyl isopeptide structures were incorporated. After ligation by the thioester method and the introduction of the palmitoyl groups, all the protecting groups were removed and the isopeptide structures were converted into the native peptide bond. Finally, the obtained polypeptide was successfully inserted into bicelles, thus showing the success of the synthesis.

Chemical synthesis of the ubiquitinated form of histone H3 and its effect on DNA methyltransferase 1.

Posttranslational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitination is one such modification. We previously reported on the synthesis of ubiquitinated histone H3 with an isopeptide mimetic structure. In this report, we describe the preparation of ubiquitinated histone H3 peptides with a native isopeptide structure, which showed a slightly weaker effect on the enzymatic activity of DNA methyltransferase 1 than the previous ubiquitinated H3 peptide analogs. These findings show that a native structure is important for determining the mechanism of the function, although ubiquitinated H3 peptide analogs can mimic the role of the original ubiquitinated H3. We also report on the successful preparation of the ubiquitinated full length histone H3.

Delayed male germ cell sex-specification permits transition into embryonal carcinoma cells with features of primed pluripotency.

Testicular teratomas result from anomalies in embryonic germ cell development. In 129 inbred mice, teratoma initiation coincides with germ cell sex-specific differentiation and the mitotic-meiotic switch: XX and XY germ cells repress pluripotency, XX germ cells initiate meiosis, and XY germ cells activate male-specific differentiation and mitotic arrest. Here, we report that expression of Nanos2, a gene that is crucial to male sex specification, is delayed in teratoma-susceptible germ cells. Decreased expression of Nanos2 was found to be due, in part, to the Nanos2 allele present in 129 mice. In teratoma-susceptible germ cells, diminished expression of genes downstream of Nanos2 disrupted processes that were crucial to male germ cell differentiation. Deficiency for Nanos2 increased teratoma incidence in 129 mice and induced developmental abnormalities associated with tumor initiation in teratoma-resistant germ cells. Finally, in the absence of commitment to the male germ cell fate, we discovered that a subpopulation of teratoma-susceptible germ cells transition into embryonal carcinoma (EC) cells with primed pluripotent features. We conclude that delayed male germ cell sex-specification facilitates the transformation of germ cells with naïve pluripotent features into primed pluripotent EC cells.

Mechanism-Based Inhibitor of DNA Cytosine-5 Methyltransferase by a SN Ar Reaction with an Oligodeoxyribonucleotide Containing a 2-Amino-4-Halopyridine-C-Nucleoside.

In chromatin, 5-methylcytosine (mC), which represents the fifth nucleobase in genomic DNA, plays a role as an inducer of epigenetic changes. Tumor cells exhibit aberrant DNA methylation patterns, and inhibition of human DNA cytosine-5 methyltransferase (DNMT), which is responsible for generating mC in CpG sequences, is an effective strategy to treat various cancers. Here, we describe the design, synthesis, and evaluation of the properties of 2-amino-4-halopyridine-C-nucleosides (dX P) and oligodeoxyribonucleotides (ODNs) containing dX P as a novel mechanism-based inhibitor of DNMTs. The designed ODN containing X PpG forms a complex with DNMTs by covalent bonding through a nucleophilic aromatic substitution (SN Ar) reaction, and its cell proliferation activity is investigated. This study suggests that dX P in a CpG sequence of DNA could serve as a potential nucleic acid drug lead in cancer chemotherapy and a useful chemical probe for studies of epigenetics. Our molecular design using a SN Ar reaction would be useful for DNMTs and other protein-DNA interactions.

Synthesis of ubiquitylated histone H3 using a thiirane linker for chemical ligation.

Post-translational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitin modification is one such modification. We previously reported on the use of a thiirane linker to introduce a 1,2-aminothiol moiety at a cysteine residue for native chemical ligation with peptide thioesters, which permitted isopeptide mimetics to be produced. In this report, we describe the preparation of the ubiquitylated full length histone H3 at the 18 position and the construction of tetranucleosomes with recombinant histones H2A, H2B, H4, and DNA, which are slightly more stable than those that are prepared without ubiquitin modification. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

5-Hydroxymethylcytosine Marks Sites of DNA Damage and Promotes Genome Stability.

5-hydroxymethylcytosine (5hmC) is a DNA base created during active DNA demethylation by the recently discovered TET enzymes. 5hmC has essential roles in gene expression and differentiation. Here, we demonstrate that 5hmC also localizes to sites of DNA damage and repair. 5hmC accumulates at damage foci induced by aphidicolin and microirradiation and colocalizes with major DNA damage response proteins 53BP1 and γH2AX, revealing 5hmC as an epigenetic marker of DNA damage. Deficiency for the TET enzymes eliminates damage-induced 5hmC accumulation and elicits chromosome segregation defects in response to replication stress. Our results indicate that the TET enzymes and 5hmC play essential roles in ensuring genome integrity.

A novel method to analyze 5-hydroxymethylcytosine in CpG sequences using maintenance DNA methyltransferase, DNMT1.

Hydroxymethylcytosine has been shown to be involved in DNA demethylation and gene expression. Although methods to determine the position of hydroxymethylcytosine at single-base resolution have been reported, these methods involve some difficulties. Here, we report a simple method to analyze hydroxymethylcytosine in the CpG sequence utilizing the maintenance DNA methylation activity of DNMT1, which selectively methylates hemi-methylated but not hemi-hydroxymethylated CpG sequences. The method enables monitoring of the dynamics of the hydroxymethylation state of a specific genome site.

Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block.

The post-translational modification of histones plays an important role in gene expression. We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal Cys residue of the C-terminal building block that is introduced for purposes of chemical ligation unprotected. To this end, a phenacyl group was successfully utilized to protect endogenous Cys residue(s), and the recombinant peptide was ligated with a peptide containing a Cys-Pro ester (CPE) sequence as a thioester precursor. Using this approach it was possible to prepare all of the core histones H2A, H2B, H3 and H4 with any modifications. The resulting proteins could then be used to prepare a core histone library of proteins that have been post-translationally modified.

NMR characterization of the interaction of the endonuclease domain of MutL with divalent metal ions and ATP.

MutL is a multi-domain protein comprising an N-terminal ATPase domain (NTD) and C-terminal dimerization domain (CTD), connected with flexible linker regions, that plays a key role in DNA mismatch repair. To expand understanding of the regulation mechanism underlying MutL endonuclease activity, our NMR-based study investigated interactions between the CTD of MutL, derived from the hyperthermophilic bacterium Aquifex aeolicus (aqMutL-CTD), and putative binding molecules. Chemical shift perturbation analysis with the model structure of aqMutL-CTD and circular dichroism results revealed that tight Zn(2+) binding increased thermal stability without changing secondary structures to function at high temperatures. Peak intensity analysis exploiting the paramagnetic relaxation enhancement effect indicated the binding site for Mn(2+), which shared binding sites for Zn(2+). The coexistence of these two metal ions appears to be important for the function of MutL. Chemical shift perturbation analysis revealed a novel ATP binding site in aqMutL-CTD. A docking simulation incorporating the chemical shift perturbation data provided a putative scheme for the intermolecular interactions between aqMutL-CTD and ATP. We proposed a simple and understandable mechanical model for the regulation of MutL endonuclease activity in MMR based on the relative concentrations of ATP and CTD through ATP binding-regulated interdomain interactions between CTD and NTD.

Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1).

Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291-1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes.

The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1delta/epsilon.

Dnmt1 (DNA methyltansferase 1) is an enzyme that recognizes and methylates hemimethylated DNA during DNA replication to maintain methylation patterns. The N-terminal region of Dnmt1 is known to form an independent domain structure that interacts with various regulatory proteins and DNA. In the present study, we investigated protein kinases in the mouse brain that could bind and phosphorylate the N-terminal regulatory domain of Dnmt1. A protein fraction containing protein kinase activity for phosphorylation of Dnmt1(1-290) was prepared using Dnmt1(1-290)-affinity, DNA-cellulose and gel-filtration columns. When the proteins in this fraction were analysed by LC-MS/MS (liquid chromatography tandem MS), CK1delta/epsilon (casein kinase 1delta/epsilon) was the only protein kinase identified. Recombinant CK1delta/epsilon was found to bind to the N-terminal domain of Dnmt1 and significantly phosphorylated this domain, especially in the presence of DNA. Phosphorylation analyses using various truncation and point mutants of Dnmt1 revealed that the major priming site phosphorylated by CK1delta/epsilon was Ser146, and that subsequent phosphorylation at other sites may occur after phosphorylation of the priming site. When the DNA-binding activity of phosphorylated Dnmt1 was compared with that of the non-phosphorylated form, phosphorylation of Dnmt1 was found to decrease the affinity for DNA. These results suggest that CK1delta/epsilon binds to and phosphorylates the N-terminal domain of Dnmt1 and regulates Dnmt1 function by reducing the DNA-binding activity.

Abnormal DNA methyltransferase expression in mouse germline stem cells results in spermatogenic defects.

Although spermatogonial stem cells (SSCs) are committed to spermatogenesis, they may also convert to an embryonic stem cell-like pluripotent state at a low frequency. Because changes in DNA methylation patterns are associated with this conversion, we examined the effect of manipulating DNA methyltransferase (Dnmt) expression on the fate of cultured SSCs, germline stem (GS) cells. Dnmt1 knockdown induced apoptosis in GS cells, which was attenuated by the loss of Trp53. In contrast, GS cells proliferated normally in vitro after Dnmt3a/Dnmt3b ablation or during Dnmt3l overexpression. However, Dnmt3a/Dnmt3b double-mutant cells showed hypomethylation in the SineB1 repetitive sequence, and Dnmt3l-overexpressing cells showed hypermethylation in major and minor satellite sequences; neither cell type formed teratomas and completed spermatogenesis following transplantation into the seminiferous tubules. Although genetic manipulation did not increase the conversion of GS cells to a pluripotent state, these results underscore the important role of DNMTs in survival and spermatogenic differentiation in SSCs.

Distinct DNA methylation activity of Dnmt3a and Dnmt3b towards naked and nucleosomal DNA.

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. De novo type DNA methyltransferases Dnmt3a and Dnmt3b are responsible for creating DNA methylation patterns during embryogenesis and in germ cells. Although their in vitro DNA methylation properties are similar, Dnmt3a and Dnmt3b methylate different genomic DNA regions in vivo. In the present study, we have examined the DNA methylation activity of Dnmt3a and Dnmt3b towards nucleosomes reconstituted from recombinant histones and DNAs, and compared it to that of the corresponding naked DNAs. Dnmt3a showed higher DNA methylation activity than Dnmt3b towards naked DNA and the naked part of nucleosomal DNA. On the other hand, Dnmt3a scarcely methylated the DNA within the nucleosome core region, while Dnmt3b significantly did, although the activity was low. We propose that the preferential DNA methylation activity of Dnmt3a towards the naked part of nucleosomal DNA and the significant methylation activity of Dnmt3b towards the nucleosome core region contribute to their distinct methylation of genomic DNA in vivo.

Expression of Dnmt3b in mouse hematopoietic progenitor cells and spermatogonia at specific stages.

Two de novo-type DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. Dnmt3b is specifically expressed in the totipotent cells of mouse early embryos and Dnmt3a, a longer form of the two isoforms, is ubiquitously expressed in mesenchyme cells after the 10 day embryo stage [Mech. Dev. 118 (2002) 187]. In the present study, we demonstrated that Dnmt3b was expressed in the nuclei of specific cells in certain tissues after the 10 day embryo stage. In fetal liver, dorsal aorta and portal vein, Dnmt3b was expressed in cells expressing CD34, indicating that the cells were hematopoietic progenitor cells. However, Dnmt3b was not expressed in the hematopoietic progenitor cells in yolk sac blood islands at 8 day embryo stage and in adult bone marrow cells. Dnmt3b was also expressed in type-A spermatogonia after birth. Dnmt3b was expressed not only in the totipotent stem cells but also in the progenitor cells the direction of differentiation of which had been already determined. On the other hand, the long form of Dnmt3a was not expressed in these hematopoietic progenitor cells in fetal liver or type-A spermatogonia, but was expressed in hepatocytes in fetal liver and type-B spermatogonia. While Dnmt3b was distributed in both the heterochromatin and euchromatin regions, Dnmt3a was specifically localized to the euchromatin region.

Cloning and characterization of a novel Ca2+/calmodulin-dependent protein kinase I homologue in Xenopus laevis.

In order to investigate protein kinases expressed in the different developmental stages of Xenopus laevis, recently developed expression cloning was carried out. When two different expression libraries, Xenopus oocyte and Xenopus head (embryonic stage 28/30) cDNA libraries, were screened by kinase-specific monoclonal antibodies, cDNA clones for various known and novel protein serine/threonine kinases (Ser/Thr kinases) were isolated. In addition to well-characterized Ser/Thr kinases, one cDNA clone for a putative kinase was isolated from the Xenopus head library. The sequence of the open reading frame of the cDNA encoded a protein of 337 amino acid residues with a predicted molecular weight of 38,404. Since the deduced animo acid sequence of this protein was 75% identical to that of rat Ca(2+)/calmodulin-dependent protein kinase I (CaMKI), it was designated as CaMKIx. Although recombinant CaMKIx expressed in Escherichia coli showed no protein kinase activity against syntide-2, a synthetic peptide substrate, it was activated when phosphorylated by mouse Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha). Activated CaMKIx significantly phosphorylated various proteins including synapsin I, histones, and myelin basic protein. CaMKIx could not be detected in the early stages of embryogenesis, but was detected in late embryos of stages 37/38 and thereafter when examined by Western blotting using a specific antibody. This kinase was found to be highly expressed in adult brain and heart, and an upstream kinase that could activate CaMKIx was detected in these tissues. These results suggest that CaMKIx plays some critical role in the late stages of embryogenesis of Xenopus laevis.

Distinct enzymatic properties of recombinant mouse DNA methyltransferases Dnmt3a and Dnmt3b.

Recombinant mouse Dnmt3a and Dnmt3b were expressed in sf9 cells and purified to near homogeneity. The purified Dnmt3a and Dnmt3b gave specific activities of 1.8 +/- 0.3 and 1.3 +/- 0.1 mol/h/mol enzyme towards poly(dGdC)-poly(dGdC), respectively, which were the highest among those reported. Dnmt3a or Dnmt3b showed similar K(m) values towards poly(dIdC)-poly(dIdC) and poly(dGdC)-poly(dGdC). The K(m) values for S-adenosyl-L-methionine were not affected by the methyl-group acceptors, poly(dI-dC)-poly(dIdC) and poly(dG-dC)-poly(dGdC). The results indicate that the enzymes are de novo-type DNA methyltransferases. Dnmt3a and Dnmt3b activities were inhibited by Mn(2+) and Ni(2+) and showed broad pH optima around neutral pH. Both enzymes were susceptible to sodium ions, which inhibited their activity at around physiological ionic strength. However, Dnmt3a was fully active at physiological potassium concentration, but Dnmt3b was not. Using designed oligonucleotides for the analysis of cytosine methylation, we demonstrated that, in addition to CpG, Dnmt3a methylated CpA but not CpT and CpC, and that Dnmt3b methylated CpA and CpT but scarcely CpC. The relative activity of Dnmt3b towards nonCpG sequences was higher than that of Dnmt3a. These differences in enzymatic properties of Dnmt3a and Dnmt3b may contribute to the distinct functions of these enzymes in vivo.

Monoclonal antibody against dnmt1 arrests the cell division of xenopus early-stage embryos.

DNA methylation plays a crucial role in embryogenesis, and Dnmt1 is known to be a key enzyme in the maintenance of DNA methylation. Dnmt1 is highly accumulated in mature oocytes and eggs. To analyze the function of the maternally accumulated Dnmt1, we injected monoclonal antibodies that specifically recognize the amino terminus of Xenopus Dnmt1 into Xenopus laevis embryos. The monoclonal antibodies inhibited the cell division of the embryos before the midblastula transition. Monoclonal antibody neither inhibited DNA methylation activity of Dnmt1 in vitro nor affected its stability in embryos. In addition, injection of alpha-amanitin, an inhibitor of transcription, did not rescue the cell division arrest. The results suggest that the inhibition of cell division by monoclonal antibodies was due neither to the direct inhibition of DNA methylation activity of Dnmt1 nor to aberrant transcription before the midblastula transition. The morphology of chromatin of the arrested cells showed that the cell cycle was arrested at interphase. This was supported by the biochemical analysis in which the arrested cells demonstrated low histone H1 kinase activity, which indicated that the cells had not entered M phase. Dnmt1 may have an important function other than DNA methylation activity for early embryogenesis in Xenopus laevis.

Exogenous expression of mouse Dnmt3 induces apoptosis in Xenopus early embryos.

Mouse DNA (cytosine-5) methyltransferases Dnmt3a and Dnmt3b are expected to be de novo-type DNA methyltransferases. In the present study, we found that exogenously expressed mouse Dnmt3a or Dnmt3b induced abnormal cell clusters at the gastrulation stage in Xenopus embryos. The abnormal cells were judged to be apoptotic from the positive staining with the TdT dUTP nucleotide end-labeling method and the rescue by hBcl-x(L), a Bcl-2 homologue. On the other hand, neither bacterial DNA (cytosine-5) methyltransferase nor Dnmt3b3, one of the three isoforms of Dnmt3b that has no DNA methylation activity, induced apoptosis. In addition, mutant Dnmt3a and the other two Dnmt3b isoforms, Dnmt3b1 and Dnmt3b2, which have no DNA methylation activity due to a change of the cysteine residue in the catalytic center to an alanine residue, retained the ability to induce apoptosis. This indicates that the apoptosis was not induced by DNA methylation activity. The domain of Dnmt3b1 (3b2) responsible for the apoptosis is the catalytic domain in the carboxyl-terminal half.