A Sensitive Electrochemical Biosensor for Detection of Histone Deacetylase Activity Using an Acetylated Peptide

A sensitive electrochemical biosensor was developed for activity detection of histone deacetylase sirtuin2 (SIRT2) using an acetylated peptide substrate. This substrate could be recognized by anti‐acetylated peptide antibody, which could be detected using secondary antibody conjugated alkaline phosphatase which provided an amplified electrochemical signal. In the presence of SIRT2, the substrate was deacetylated, resulting in a decreased electrochemical signal that was correlated to the concentration of SIRT2. Under optimized conditions, the biosensor exhibited a wide linear range from 1 nM to 500 nM with a detection limit of 0.1 nM. The proposed biosensor was also used for detection of SIRT2 inhibitor.     

Azalysine analogues as probes for protein lysine deacetylation and demethylation

Reversible lysine acetylation and methylation regulate the function of a wide variety of proteins, including histones. Here, we have synthesized azalysine-containing peptides in acetylated and unacetylated forms as chemical probes of the histone deacetylases (HDAC8, Sir2Tm, and SIRT1) and the histone demethylase, LSD1. We have shown that the acetyl-azalysine modification is a fairly efficient substrate for the sirtuins, but a weaker substrate for HDAC8, a classical HDAC. In addition to deacetylation by sirtuins, the acetyl-azalysine analogue generates a novel ADP-ribose adduct that was characterized by mass spectrometry, Western blot analysis, and nuclear magnetic resonance spectroscopy. This peptide-ADP-ribose adduct is proposed to correspond to a derailed reaction intermediate, providing unique evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed in the course of sirtuin catalyzed deacetylation. An unacetylated azalysine-containing H3 peptide proved to be a potent inhibitor of the LSD1 demethylase, forming an FAD adduct characteristic of previously reported related structures, providing a new chemical probe for mechanistic analysis.     

HDAC对接研究:苯甲酰胺类抑制剂结合方式推测

通过计算机模拟的对接过程研究，发现了MS-275— 一种苯甲酰胺类的组蛋白去乙酰酶（HDAC）抑制剂与酶的可能的全新结合方式.这种结合方式与已经阐明的组蛋白去乙酰酶类似蛋白（HDLP）与曲古柳菌素A(trichostatin A, TSA)和suberoylanilide hydroxamic acid(SAHA)形成的复合物晶体结构中配体与酶的作用方式完全不同.从对接结果看，MS-275的作用靶点在酶活性口袋的最狭窄部位，而不是直接作用于锌离子.这似乎能够解释MS-275的低毒性特点，并且为设计和筛选全新的HDAC抑制剂提供了新思路.     

Analysis of human histone H4 by capillary electrophoresis in a pullulan-coated capillary,LC-ESI-MS and MALDI-TOF-MS

The object of the present study was the analysis of the human histone H4 (a core histone) in order to evaluate the state of its acetylation. Capillary electrophoresis (CE) using a pullulan-coated capillary provides a rapid and efficient approach to the separation of monoacetylated, diacetylated and triacetylated H4 isoforms from human cells. By using a simple running buffer of 100 mM triethanolamine-phosphate solution at pH 2.5 and exploiting the effectiveness of pullulan-based coverage in preventing adsorptive phenomena, the separation of the differently acetylated isoforms was achieved in less than 15 min with high efficiency and reproducibility. The proposed method was for the first time applied in the analysis of histone H4 fractions obtained from cell lines treated with different histone deacetylase (HDAC) inhibitors, used as potential anticancer drugs. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis demonstrated that the acetylation occurred in the histone H4 tail, whereas the CE separation allowed for a fast determination of the percentages of H4 acetylated isoforms in real samples; the results were in agreement with those obtained from liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) analysis. Therefore, the proposed CE method is a useful complementary support to the hyphenated techniques for the rapid monitoring of the activity of HDAC inhibitors.     

Europium(III) chelate-dyed nanoparticles as donors in a homogeneous proximity-based immunoassay for estradiol

Nanoparticles containing thousands of fluorescent europium(III) chelates have a very high specific activity compared to traditional lanthanide chelate labels. It can be assumed that if these particles are used in a homogeneous assay as donors, multiple chelates can excite a single acceptor in turns and the energy transfer to the acceptor is increased. The principle was employed in an immunoassay using luminescent resonance energy transfer from a long lifetime europium(III) chelate-dyed nanoparticle to a short lifetime, near-infrared fluorescent molecule. Due to energy transfer fluorescence lifetime of the sensitised emission was prolonged and fluorescence could be measured using a time-resolved detection.A competitive homogeneous immunoassay for estradiol was created using 92 nm europium(III) chelate-dyed nanoparticle coated with 17β-estradiol specific recombinant antibody Fab fragments as a donor and estradiol conjugated with near-infrared dye AlexaFluor 680 as an acceptor. The density of Fab fragments on the surface of the particle influenced the sensitivity of the immunoassay. The optimal Fab density was reached when the entire surface of the particle participated in the energy transfer, but the areas where the energy was transferred to a single acceptor, did not overlap. We were able to detect estradiol concentrations down to 70 pmol l⁻¹ (3×SD of a standard containing 0 nmol l⁻¹ of E2) using a 96-well platform. In this study we demonstrated that nanoparticles containing lanthanide chelates could be used as efficient donors in homogeneous assays.     

金纳米颗粒表面能量转移及巯基化合物的检测

二氧化硅稳定的金纳米颗粒(Au-SiO₂)与罗丹明B之间发生表面能量转移,使罗丹明B荧光猝灭。 金纳米颗粒对罗丹明B的Stern-Volmer猝灭常数为4.3×10³ L/mol。 当荧光猝灭的混合体系中加入巯基化合物时,巯基化合物与金纳米颗粒发生强相互作用阻断罗丹明B-金纳米颗粒之间的能量转移,罗丹明B荧光恢复。 基于罗丹明B-Au-SiO₂体系对巯基化合物的单一响应,建立了一种简单快速检测巯基化合物的方法;并且由于二氧化硅对金纳米颗粒的稳定作用,金纳米颗粒成为一种可以回收利用的检测探针。     

一种香豆素荧光标记多肽的合成及应用

Sirtuin蛋白是一类称为依赖烟酰胺腺嘌呤二核苷酸（NAD）的组蛋白去乙酰化酶,共有7个成员,均是潜在的疾病治疗靶点。 然而,目前的荧光筛选方法,只适用于SIRT1~SIRT3。 因此,根据SIRT5的新酶活,设计、合成了针对SIRT5的荧光标记多肽（ISGASE(SuK) AMC）,并通过LC-MS和荧光检测证明了该荧光标记多肽能应用于SIRT5的活性筛选。     

Excitation Energy Transfer from Semi‐Conducting Polymer Nanoparticles to Surface‐Bound Fluorescent Dyes

Summary: The first examples of the dye‐coated semi‐conducting polymer nanoparticles as well as experiments to demonstrate the excitation energy transfer from the excited chromophor of the nanoparticle to the fluorescent dye are described. We have demonstrated that the blue fluorescence of the dye‐coated polyfluorene nanoparticles is only slightly quenched after dye deposition. However, a new emission band of the surface‐bound dye (Rhodamine 6G or Rhodamine TM) appears in the wavelength region of 530–600 nm. These results clearly indicate an effective excitation energy transfer from the excited PF chromophores to the fluorescent dye.

Emission spectra of PF2/6 nanoparticle dispersion and of Rhodamine 6G‐coated nanoparticle dispersion.     

Design,Synthesis and Biological Evaluation of Novel Selective Thiol-based Histone Deacetylase(HDAC) VI InhibitorsBearing Indeno[1,2-c]pyrazole or Benzoindazole Scaffold

A series of thiol-based indeno[1,2-c]pyrazoles and benzoindazole compounds was designed and synthesized according to the structural specificity of histone deacetylase VI(HDAC6) and the structural characteristics of HDAC inhibitors. The inhibitory activities of the target compounds against HDAC6 and HDAC1 were screened by fluorescence analysis. Most of the target compounds showed moderate inhibitory activity against HDAC6(IC₅₀=44—598 nmol/L). Among them, compound A-4 displayed the highest selectivity against HDAC6 and similar inhibitory activity(IC₅₀=44 nmol/L) to that of the positive drug SAHA(IC₅₀=41 nmol/L) against HDAC6.     

Sensitive turn-on fluorescent detection of melamine based on fluorescence resonance energy transfer

We here report a novel fluorescent method for the detection of melamine based on the high fluorescence quenching ability of gold nanoparticles. The fluorescence was significantly quenched via fluorescence resonance energy transfer when fluorescein molecules were attached to the surface of gold nanoparticles by electrostatic interaction. Upon addition of melamine, the fluorescence was enhanced due to the competitive adsorption of gold nanoparticles between melamine and fluorescein. Under the optimum conditions, the fluorescence enhancement efficiency [(I-I(0))/I(0)] showed a linear relationship with the concentration of melamine in the range of 1.0 × 10(-7) mol L(-1)~4.0 × 10(-6) mol L(-1), and the detection limit was calculated to be 1.0 × 10(-9) mol L(-1). The proposed method showed several advantages such as high sensitivity, short analysis time, low cost and ease of operation.     

信号肽功能化二氧化硅纳米颗粒的线粒体靶向作用研究

以N-(p-Maleimidophenyl)isocyanate(PMPI)为交联剂, 将线粒体信号肽分子共价修饰到二氧化硅荧光纳米颗粒表面, 构建线粒体信号肽功能化二氧化硅荧光纳米颗粒. 采用荧光分光光度计、Zeta电位仪以及透射电子显微镜对修饰前后的二氧化硅纳米颗粒进行了表征. 结果表明, 信号肽可被成功修饰在纳米颗粒表面, 并且纳米颗粒粒径在信号肽分子修饰前后没有发生明显变化. 以分离纯化的细胞核作为对照, 采用流式细胞术考察了信号肽功能化二氧化硅荧光纳米颗粒与分离纯化后的线粒体的相互作用. 结果表明, 线粒体信号肽修饰到二氧化硅纳米颗粒表面后依然保持良好的生物活性, 能够介导二氧化硅纳米颗粒特异性识别及结合分离纯化的线粒体, 从而为线粒体监测及其功能调控研究提供了新的思路.     

A DNA hybridization detection based on fluorescence resonance energy transfer between dye-doped core-shell silica nanoparticles and gold nanoparticles

A novel and efficient method to evaluate the DNA hybridization based on a fluorescence resonance energy transfer (FRET) system, with fluorescein isothiocyanate (FITC)-doped fluorescent silica nanoparticles (SiNPs) as donor and gold nanoparticles (AuNPs) as acceptor, has been reported. The strategy for specific DNA sequence detecting is based on DNA hybridization event, which is detected via excitation of SiNPs-oligonucleotide conjugates and energy transfer to AuNPs-oligonucleotide conjugates. The proximity required for FRET arises when the SiNPs-oligonucleotide conjugates hybridize with partly complementary AuNPs-oligonucleotide conjugates, resulting in the fluorescence quenching of donors, SiNPs-oligonucleotide conjugates, and the formation of a weakly fluorescent complex, SiNPs-dsDNA-AuNPs. Upon the addition of the target DNA sequence to SiNPs-dsDNA-AuNPs complex, the fluorescence restores (turn-on). Based on the restored fluorescence, a homogeneous assay for the target DNA is proposed. Our results have shown that the linear range for target DNA detection is 0-35.0 nM with a detection limit (3σ) of 3.0 picomole. Compared with FITC-dsDNA-AuNPs probe system, the sensitivity of the proposed probe system for target DNA detection is increased by a factor of 3.4-fold.     

Facile synthesis of colorimetric histone deacetylase substrates

Here we report a simple procedure for generating colorimetric histone deacetylase (HDAC) substrates by solid-phase peptide synthesis based on racemization-free couplings of amino acid chlorides. We demonstrate the applicability of these substrates in HDAC assays.     

Nucleic acids induced peptide-based AIE nanoparticles for fast cell imaging

Herein, we utilized nucleic acids induced peptide co-assembly strategy to develop novel nucleic acids induced peptide-based AIE (NIP-AIE) nanoparticles. Strong fluorescent of AIE could be observed when a little amount of nucleic acids was added into the peptide solution, and the intensity could be regulated by the concentration of nucleic acids. This AIE nanoparticle with good biocompatibility could achieve fast cell imaging. It is also proved that the fluorescence intensity of AIE decreased with time, which indicates that the reducible cross-linkers of Wpc peptide by GSH and nanoparticles gradually disintegrate in cell. Based on the different of AIE fluorescence signals which regulated by the formation and disintegration of nanoparticles, this AIE system is expected to be used for real-time monitoring of drug release from peptide-based nano carriers in vivo or in vitro, and may provide a new platform for the construction of other organic AIE nanoparticles.     

A Versatile Strategy for Screening Custom-Designed Warhead-Armed Cyclic Peptide Inhibitors

Demand for peptide-based pharmaceuticals has been steadily increasing, but only limited success has been achieved to date. To expedite peptide-based drug discovery, we developed a general scheme for cell-based screening of cyclic peptide inhibitors armed with a user-designed warhead. We combined unnatural amino acid incorporation and split intein-mediated peptide cyclization techniques and integrated a yeast-based colorimetric screening assay to generate a new scheme that we call the custom-designed warhead-armed cyclic peptide screening platform (CWCPS). This strategy successfully discovered a potent inhibitor, CY5-6Q, that targets human histone deacetylase 8 (HDAC8) with a K_D value of 15 nM. This approach can be a versatile and general platform for discovering cyclic peptide inhibitors.     

Functional gold nanoparticle-peptide complexes as cell-targeting agents

In this paper, we report a novel approach using peptide CALNN and its derivative CALNNGGRRRRRRRR (CALNNR(8)) to functionalize gold nanoparticles for intracellular component targeting. The translocation is effected by the nanoparticle diameter and CALNNR(8) surface coverage. The intracellular distributions of the complexes are change from the cellular nucleus to the endoplasmic reticulum by increasing the density of CALNNR(8) at a constant nanoparticle diameter. Additionally, increasing the nanoparticle diameter at a constant density of CALNNR(8) leads to less cellular internalization. These translocations of the complexes cause unique colorimetric expressions of the cell structure. The cell viability is affected by the internalized gold nanoparticle-peptide complexes in terms of quantities of particles per cell. In addition, the intracellular distribution of the fluorescence quenching is investigated by a fluorescent confocal scanning laser microscopy, which also gives further evidence of intracellular distribution of the gold nanoparticle-peptide complexes.     

Genetically encoded fluorescent reporters of histone methylation in living cells

We report the design and characterization of two genetically encoded fluorescent reporters of histone protein methylation. The reporters are four-part chimeric proteins consisting of a substrate peptide from the N-terminus of histone H3 fused to a chromodomain (a natural methyllysine-specific recognition domain), sandwiched between a fluorescence resonance energy transfer (FRET)-capable pair of fluorophores, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Enzymatic methylation by a methyltransferase induces complexation of the methylated substrate peptide to the chromodomain, changing the FRET level between the flanking CFP and YFP domains. Reporters developed using the chromodomains from HP1 and Polycomb respond to enzymatic methylation at the lysine 9 and lysine 27 positions of histone H3, respectively, giving 60% and 28% YFP/CFP emission ratio increases in vitro or in single living cells. These reporters should be useful for studying gene silencing and X-chromosome inactivation with high spatial and temporal resolution in intact cells and may also aid in the search for conjectured histone demethylase activity.     

Synthesis of gold–poly(methyl methacrylate) core–shell nanoparticles by surface‐confined atom transfer radical polymerization at elevated temperature

Surface‐confined atom transfer radical polymerization was used to prepare gold nanoparticle–poly(methyl methacrylate) core–shell particles at elevated temperature. First, gold nanoparticles were prepared by the one‐pot borohydride reduction of tetrachloroaurate in the presence of 11‐mercapto‐1‐undecanol (MUD). MUD‐capped gold nanoparticles were then exchanged with 3‐mercaptopropyltrimethoxysilane (MPS) to prepare a self‐assembled monolayer (SAM) of MPS on the gold nanoparticle surfaces and subsequently hydrolyzed with hydrochloric acid. The extent of exchange of MUD with MPS was determined by NMR. The resulting crosslinked silica‐primer layer stabilized the SAM of MPS and was allowed to react with the initiator [(chloromethyl)phenylethyl] trimethoxysilane. Atom transfer radical polymerization was conducted on the Cl‐terminated gold nanoparticles with the CuCl/2,2′‐bipyridyl catalyst system at elevated temperature. The rates of polymerization with the initiator‐modified gold nanoparticles exhibited first‐order kinetics with respect to the monomer, and the number‐average molecular weight of the cleaved graft polymer increased linearly with the monomer conversion. The presence of the polymer on the gold nanoparticle surface was identified by Fourier transform infrared spectroscopy and transmission electron microscopy. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 3631–3642, 2005