Profiling of Substrates for Zinc-dependent Lysine Deacylase Enzymes: HDAC3 Exhibits Decrotonylase Activity In Vitro

Systematic screening of the activities of the eleven human zinc-dependent lysine deacylases against a series of fluorogenic substrates as well as kinetic evaluation revealed substrates for screenings of histone deacetylases HDAC10 and HDAC11 at reasonably low enzyme concentrations. Furthermore, HDAC3 in complex with nuclear receptor corepressor?1 (HDAC3-NCoR1) was shown to harbor decrotonylase activity in?vitro.     

水稻组蛋白单泛素化连接酶基因(OsHUB1)的克隆及其表达分析

对模式植物拟南芥的研究发现,AtHUB1基因参与调控植物对灰霉病的抗性.为了研究水稻的抗病机理,笔者利用同源搜索从水稻数据库中获得了目标基因的序列,并通过RT-PCR技术从水稻中克隆了组蛋白单泛素化连接酶基因,命名为OsHUB1.该片段包含1个2 655 bp的开放阅读框,编码了1个885氨基酸的多肽.与NCBI数据库的比对结果表明:OsHUB1的氨基酸序列与短柄草(Brachypodium distachyon)、葡萄(Vitis vinifera)、杨树(Populus tomentosa)、大豆(Glycine max)和拟南芥(Arabidopsis thaliana)的泛素E3连接酶的同源性分别为78%,68%,68%,67%和62%.这些不同来源的组蛋白单泛素化连接酶都含有一个高度保守的RING指结构域.半定量表达分析结果表明:OsHUB1基因能够被水杨酸(SA)和茉莉酸(JA)诱导表达,说明OsHUB1基因可能通过SA和JA介导的信号转导途径参与水稻的抗病反应.     

Etoposide interferes with the process of chromatin condensation during alga Chara vulgaris spermiogenesis

DNA topoisomerase II plays an essential role in animal spermiogenesis, where changes of chromatin structure are connected with appearance of transient DNA breaks. Such topo II activity can be curtailed by inhibitors such as etoposide and suramine. The aim of the present study was to investigate, for the first time, the effect of etoposide on spermatid chromatin remodeling in the green alga Chara vulgaris. This inhibitor prolonged the early spermiogenesis stages and blocked the formation of the phosphorylated form of histone H2AX at stages VI–VII. The lack of transient DSBs at these stages impairs the elimination of supercoils containing nucleosomes which lead to disturbances in nucleoprotein exchange and the pattern of spermatid chromatin fibrils at stages VI–VIII. Immunofluorescent and ultrastructural observations revealed that during C. vulgaris spermiogenesis topo II played an important role similar to that in mammals. Some corresponding features had been pointed out before, the present studies showed further similarities.     

Development and validation of a LC‐MS assay for the quantification of ikh12 a novel anti‐tumor candidate in rat plasma and tissues and its application in a pharmacokinetic study

IKH12 is a novel histone deacetylase 6 selective inhibitor. A rapid and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of IKH12 in rat plasma and tissue with kendine 91 as internal standard (IS). The samples were prepared by liquid–liquid extraction with tert‐butyl methyl ether. The chromatographic separation was accomplished by using a Zorbax Extend C₁₈ 4.6 × 150 mm, 5 µm column, with a mobile phase consisting of methanol and 0.1% formic acid (75:25 v/v). Multiple reaction monitoring, using electrospray ionization in positive ion mode, was employed to quantitatively detect IKH12 and IS. The monitored transitions were set at m/z 418 → 252 and 444 → 169 for IKH12 and kendine 91, respectively. The calibration curve was linear over the concentration range 2–1000 ng mL^?1. The intra‐ and inter‐assay precision and accuracy of the quality controls and the limit of quantification were satisfactory in all cases (according to European Medicines Agency guidelines). Stability studies showed that plasma samples were stable in the chromatography rack for 24 h and at ?80 °C for 2 months and also after three freeze–thaw cycles. This method was successfully applied to a pharmacokinetic study of IKH12 in rat. Copyright © 2015 John Wiley & Sons, Ltd.     

Homology modeling,force field design,and free energy simulation studies to optimize the activities of histone deacetylase inhibitors

As an effort to develop therapeutics for cancer treatments, a number of effective histone deacetylase inhibitors with structural diversity have been discovered. To gain insight into optimizing the activity of an identified lead compound, a computational protocol sequentially involving homology modeling, docking experiments, molecular dynamics simulation, and free energy perturbation calculations was applied for rationalizing the relative activities of known histone deacetylase inhibitors. With the newly developed force field parameters for the coordination environment of the catalytic zinc ion in hand, the computational strategy proved to be successful in predicting the rank orders for 12 derivatives of three hydroxamate-based inhibitor scaffolds with indole amide, pyrrole, and sulfonamide moieties. The results showed that the free energy of an inhibitor in aqueous solution should be an important factor in determining the binding free energy. Hence, in order to enhance the inhibitory activity by adding or substituting a chemical group, the increased stabilization in solution due to the structural changes must be overcome by a stronger enzyme-inhibitor interaction. It was also found that to optimize inhibitor potency, the hydrophobic head of an inhibitor should be elongated or enlarged so that it can interact with Pro29 and His28 that are components of the flexible loop at the top of the active site.     

Study the effects of metallic ions on the combination of DNA and histones with molecular combing technique

The effects of monovalent (Na^ , K^ ) and divalent (Mg^2 , Ca^2 , Mn^2 ) ions on the interaction between DNA and histone are studied using the molecular combing technique. λ-DNA molecules and DNA-histone complexes incubated with metal cations (Na^ , K^ , Mg^2 , Ca^2 , Mn^2 ) are stretched on hydrophobic surfaces, and directly observed by fluorescence microscopy. The results indicate that when these cations are added into the DNA solution, the fluorescence intensities of the stained DNA are reduced differently. The monovalent cations (Na^ , K^ ) inhibit binding of histone to DNA. The divalent cations (Mg^2 , Ca^2 , Mn^2 ) enhance significantly the binding of histone to DNA and the binding of the DNA-histone complex to the hydrophobic surface. Mn^2 also induces condensation and aggregation of the DNA- histone complex.     

Study the effects of divalent metallic ions on the combination of DNA and histones with fluorescence anisotropy assays

The effects of divalent ions(Mn2 ,Mg2 ,Ca2 ) on the interaction between DNA and histone are studied using a fluorescence anisotropy assay. Fluorescence anisotropies of DNA and DNA-histone in the presence of divalent ions(Mn2 ,Mg2 ,Ca2 ) are measured. The results indicate that histone reduces the fluorescence anisotropy of lambda DNA while the divalent ions(Mn2 ,Mg2 ,Ca2 ) significantly enhance the fluorescence anisotropy. Compared to the case of DNA incubated with histone alone,there are more histones binding to DNA when divalent ion,histone and DNA are incubated together. We also find that Mn2 makes the DNA-histone complexes more condensed than the other ions do.     

组蛋白甲基化酶Set2片段调控SET结构域催化活性的探讨

以酿酒酵母为研究材料,通过体外甲基化、体内免疫共沉淀等一系列实验,研究了酵母组蛋白甲基转移酶Set2片段调控SET结构域催化活性的机制。发现将SRI 结构域敲除后（1─618片段）,仅催化产生H3K36的单甲基化,将其WW结构域、CC结构域敲除后（1─475片段）,H3K36无法被甲基化。将Set2截取到仅剩SET催化结构域（1─261片段）,H3K36又可被一甲基化和部分的二甲基化修饰。研究结果表明SET结构域的催化活性并非由Set2蛋白自身折叠调控。     

黑腹果蝇种组组蛋白基因间内含子区的分子进化

通过分析黑腹果蝇种组(Drosophila melanogasterspecies group)9个种亚组代表种和D.pseudoobscura的组蛋白基因H2A和H2B的内含子的碱基组成、替换速率、转换/颠换比、二级结构和系统发育关系等发现:整个序列长度变异范围在201 bp(ficusphila)到232 bp(takahashii)之间,替换速率为0.82,转换明显高于颠换,内含子和外显子结合区不遵循“GT-AG”和“AT-AC”模式,而是“TT-AG”模式,二级结构与系统分化关系具有相关性.我们认为组蛋白基因H2A和H2B的内含子是先起源的,在进化过程中由于承受的选择压力不同而发生了变异.