A cDNA library from the antenna of Monochamus alternatus Hope and binding properties of odorant‐binding proteins

Chemoreception is a key feature in selection of host plants by insects. We performed a preliminary functional characterization of olfactory proteins isolated from an antennal cDNA library of Monochamus alternatus. We identified four olfactory genes, including two encoding putative classic odorant‐binding proteins (OBPs) and two encoding minus‐C OBPs. We expressed two of the four OBPs, MaltOBP3 and MaltOBP5, in a bacterial system and assessed their ligand specificity by measuring the competitive binding of fluorescent probe, N‐phenyl‐1‐naph‐thylamine, in the presence of 17 volatile beetle‐ or host‐plant‐related ligands. The results indicated that although MaltOBP3 and MaltOBP5 bound a distinctly different group of competitors, both had relatively high binding affinities (K_i < 20 μm ) for certain compounds. The differences in their binding affinities towards host‐plant ligands suggest the roles of MaltOBP3 and MaltOBP5 in host‐plant selection.     

β‐Ionone as putative semiochemical suggested by ligand binding on an odorant‐binding protein of Hylamorpha elegans and electroantennographic recordings

Currently, odorant‐binding proteins (OBPs) are considered the first filter for olfactory information for insects and constitute an interesting target for pest control. Thus, an OBP (HeleOBP) from the scarab beetle Hylamorpha elegans (Burmeister) was identified, and ligand‐binding assays based on fluorescence and in silico approaches were performed, followed by a simulated binding assay. Fluorescence binding assays showed slight binding for most of the ligands tested, including host‐plant volatiles. A high binding affinity was obtained for β‐ionone, a scarab beetle‐related compound. However, the binding of its analogue α‐ionone was weaker, although it is still considered good. On the other hand, through a three‐dimensional model of HeleOBP constructed by homology, molecular docking was carried out with 29 related ligands to the beetle. Results expressed as free binding energy and fit quality (FQ) indicated strong interactions of sesquiterpenes and terpenoids (α‐ and β‐ionone) with HeleOBP as well as some aromatic compounds. Residues such as His102, Tyr105 and Tyr113 seemed to participate in the interactions previously mentioned. Both in silico scores supported the experimental affinity for the strongest ligands. Therefore, the activity of α‐ionone, β‐ionone and 2‐phenyl acetaldehyde at antennal level was studied using electroantenography (EAG). Results showed that the three ligands are electrophysiologically active. However, an aliquot of β‐ionone (represented by 3.0 ng) elicited stronger EAG responses in antennae of males than of females. Finally, the role of these ligands as potential semiochemicals for H. elegans is discussed.     

Two fatty acid‐binding proteins expressed in the intestine interact differently with endocannabinoids

Two different members of the fatty acid‐binding protein (FABP) family are found in enterocyte cells of the gastrointestinal system, namely liver‐type and intestinal fatty acid‐binding proteins (LFABP and IFABP, also called FABP1 and FABP2, respectively). Striking phenotypic differences have been observed in knockout mice for either protein, for example, high fat‐fed IFABP‐null mice remained lean, whereas LFABP‐null mice were obese, correlating with differences in food intake. This finding prompted us to investigate the role each protein plays in directing the specificity of binding to ligands involved in appetite regulation, such as fatty acid ethanolamides and related endocannabinoids. We determined the binding affinities for nine structurally related ligands using a fluorescence competition assay, revealing tighter binding to IFABP than LFABP for all ligands tested. We found that the head group of the ligand had more impact on binding affinity than the alkyl chain, with the strongest binding observed for the carboxyl group, followed by the amide, and then the glycerol ester. These trends were confirmed using two‐dimensional ¹H–¹⁵N nuclear magnetic resonance (NMR) to monitor chemical shift perturbation of the protein backbone resonances upon titration with ligand. Interestingly, the NMR data revealed that different residues of IFABP were involved in the coordination of endocannabinoids than those implicated for fatty acids, whereas the same residues of LFABP were involved for both classes of ligand. In addition, we identified residues that are uniquely affected by binding of all types of ligand to IFABP, suggesting a rationale for its tighter binding affinity compared with LFABP.     

Molecular and functional characterization of three odorant binding proteins from the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricide)

Odorant binding proteins (OBPs) act in recognizing odor molecules and their most well‐studied functions are transporting odors across the sensillum lymph to olfactory receptor neurons within the insect antennal sensillum. The adults of Grapholita molesta highly depend on olfactory cues in locating host plants and selecting oviposition sites, in which OBPs play an important role in perceiving and recognizing host plant volatiles. Exploring the physiological function of OBPs could facilitate our understanding of their importance in insects’ chemical communication. In this study, three OBP genes were cloned and named GmolOBP4, GmolOBP5, and GmolOBP10. Quantitative real‐time PCR results indicated that GmolOBP4 and GmolOBP10 were predominantly expressed in adult antennae and GmolOBP5 was expressed in multiple tissues, including head, legs, and wings in addition to antennae. The binding affinities of the three recombinant GmolOBPs (rGmolOBPs) with four sex pheromone components and twenty‐nine host plant volatiles were measured using 1‐N‐Phenyl‐naphthylamine as a fluorescence probe. The three rGmolOBPs exhibited specific binding properties to potential ligands, GmolOBP4 and GmolOBP10 bound to minor sex pheromone components, such as (Z)‐8‐dodecenyl alcohol and dodecanol, respectively. rGmolOBP4 showed intermediate binding ability with hexanal, benzyl alcohol, and pear ester, rGmolOBP5 had a weak affinity for benzaldehyde, pear ester and, methyl jasmonate, and rGmolOBP10 showed strong binding capacity toward hexanol, decanol, and α‐ocimene. We speculate that the GmolOBP4 and GmolOBP10 have dual functions in perception and recognition of host plant volatiles and sex pheromone components, while GmolOBP5 may serve other function(s).     

中华按蚊气味结合蛋白AsinOBP2的基因克隆、表达谱及与人体气味物质的结合特性分析

[目的]克隆中华按蚊Anopheles sinensis气味结合蛋白2(odorant binding protein 2,OBP2)基因AsinOBP2,分析该基因的表达及其重组蛋白与人体气味物质的结合能力.[方法]采用RT-PCR和RACE技术克隆AsinOBP2的全长cDNA序列,通过qPCR分析AsinOBP2...     

Polypyrimidine tract‐binding protein is required for the repression of gene expression by all‐trans retinoic acid

All‐trans retinoic acid is a key regulator of early development. High concentrations of retinoic acid interfere with differentiation and migration of neural crest cells. Here we report that a dinucleotide repeat in the cis‐element of Snail2 (previously known as Slug) gene plays a role in repression by all‐trans retinoic acid. We analyzed the cis‐acting regulatory regions of the Xenopus Snail2 gene, whose expression is repressed by all‐trans retinoic acid. The analysis identified a TG/CA repeat as a necessary element for the repression. By performing a yeast one‐hybrid screen, we found that a polypyrimidine tract‐binding protein (PTB), which is known to be a regulator of the alternative splicing of pre‐messenger RNA, binds to the TG/CA repeat. Overexpression and knockdown experiments for PTB in HEK293 cells and Xenopus embryos indicated that PTB is required for repression by retinoic acid. The green fluorescent protein‐PTB fusion protein was localized in the nucleus of 293T cells. In situ hybridization for PTB in Xenopus embryos showed that PTB is expressed at the regions including neural crest at the early stages. Our results indicate that PTB plays a role in the repression of gene expression by retinoic acid through binding to the TG/CA repeats.     

Fibronectin‐binding proteins of Clostridium perfringens recognize the III1‐C fragment of fibronectin

The Clostridium perfringens strain 13 genome contains two genes (fbpA, fbpB) that encode putative Fbp. Both rFbpA and rFbpB were purified and their reactivity with human serum Fn was analyzed. To determine the region of the Fn molecule recognized by rFbp, a plate binding assay using N‐terminal 70‐kDa peptide, III₁‐C peptide, and 110‐kDa peptide containing III_2–10 of Fn was performed. Both rFbp bound to the III₁‐C peptide of Fn but not to the other peptides. However, the III₁‐C fragment of Fn is known to be cryptic in serum Fn. Then, rFbp‐BP from Fn were purified by rFbp‐affinity chromatography. The yield of purified proteins was approximately 1% of the applied Fn on a protein basis. Western blotting analysis of the rFbp‐BP, using four different anti‐Fn monoclonal antibodies, revealed that the rFbp‐BP carried partial Fn antigenicity. Bindings of rFbp to rFbp‐BP were inhibited by the presence of the III₁‐C peptide, suggesting that rFbp‐BP express the III₁‐C fragment. The binding of Fn to III₁‐C was inhibited by the presence of either rFbpA or rFbpB. This result that suggests C. perfringens Fbps may inhibit the formation of Fn‐matrix in vivo.     

Synthesis and DNA‐binding study of imidazole linked thiazolidinone derivatives

A novel series of imidazole‐linked thiazolidinone hybrid molecules were designed and synthesized through a feasible synthetic protocol. The molecules were characterized with Fourier transform infrared (FT‐IR), ¹H nuclear magnetic resonance (NMR), ¹³C NMR and high‐resolution mass spectrometry (HRMS) techniques. In vitro susceptibility tests against Gram‐positive (S. aureus and B. subtilis ) and Gram‐negative bacteria (E. coli and P. aeruginosa ) gave highly promising results. The most active molecule (3e) gave a minimal inhibitory concentration (MIC) value of 3.125 μg/mL which is on par with the reference drug streptomycin. Structure–activity relationships revealed activity enhancement by nitro and chloro groups when they occupied meta position of the arylidene ring in 2‐((3‐(imidazol‐1‐yl)propyl)amino)‐5‐benzylidenethiazolidin‐4‐ones. DNA‐binding study of the most potent molecule 3e with salmon milt DNA (sm‐DNA) under simulated physiological pH was probed with UV–visible absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. These studies established that compound 3e has a strong affinity towards DNA and binds at DNA minor groove with a binding constant (K_b) 0.18 × 10² L mol^?1. Molecular docking simulations predicted strong affinity of 3e towards DNA with a binding affinity (ΔG) ‐8.5 kcal/mol. Van der Waals forces, hydrogen bonding and hydrophobic interactions were predicted as the main forces of interaction. The molecule 3e exhibited specific affinity towards adenine–thiamine base pairs. Copyright © 2016 John Wiley & Sons, Ltd.     

mRNA display selection and solid‐phase synthesis of Fc‐binding cyclic peptide affinity ligands

Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance to proteolysis, and higher affinity and specificity relative to linear peptides. Here we describe the discovery, synthesis and characterization of novel cyclic peptide affinity ligands that bind the Fc portion of human Immunoglobulin G (IgG; hFc). We generated an mRNA display library of cyclic pentapeptides wherein peptide cyclization was achieved with high yield and selectivity, using a solid‐phase crosslinking reaction between two primary amine groups, mediated by a homobifunctional linker. Subsequently, a pool of cyclic peptide binders to hFc was isolated from this library and chromatographic resins incorporating the selected cyclic peptides were prepared by on‐resin solid‐phase peptide synthesis and cyclization. Significantly, this approach results in resins that are resistant to harsh basic conditions of column cleaning and regeneration. Further studies identified a specific cyclic peptide—cyclo[Link‐M‐WFRHY‐K]—as a robust affinity ligand for purification of IgG from complex mixtures. The cyclo[Link‐M‐WFRHY‐K] resin bound selectively to the Fc fragment of IgG, with no binding to the Fab fragment, and also bound immunoglobulins from a variety of mammalian species. Notably, while the recovery of IgG using the cyclo[Link‐M‐WFRHY‐K] resin was comparable to a Protein A resin, elution of IgG could be achieved under milder conditions (pH 4 vs. pH 2.5). Thus, cyclo[Link‐M‐WFRHY‐K] is an attractive candidate for developing a cost‐effective and robust chromatographic resin to purify monoclonal antibodies (mAbs). Finally, our approach can be extended to efficiently generate and evaluate cyclic peptide affinity ligands for other targets of interest. Biotechnol. Bioeng. 2013; 110: 857–870. © 2012 Wiley Periodicals, Inc.     

The role of chemosensory protein 10 in the detection of behaviorally active compounds in brown planthopper,Nilaparvata lugens

Chemosensory proteins (CSPs) play important roles in insects’ chemoreception, although their specific functional roles have not been fully elucidated. In this study, we conducted the developmental expression patterns and competitive binding assay as well as knock‐down assay by RNA interference both in vitro and in vivo to reveal the function of NlugCSP10 from the brown planthopper (BPH), Nilaparvata lugens (Stål), a major pest in rice plants. The results showed that NlugCSP10 messenger RNA was significantly higher in males than in females and correlated to gender, development and wing forms. The fluorescence binding assays revealed that NlugCSP10 exhibited the highest binding affinity with cis‐3‐hexenyl acetate, eicosane, and (+)‐β‐pinene. Behavioral assay revealed that eicosane displayed attractant activity, while cis‐3‐hexenyl acetate, similar to (+)‐β‐pinene significantly repelled N. lugens adults. Silencing of NlugCSP10, which is responsible for cis‐3‐hexenyl acetate binding, significantly disrupted cis‐3‐hexenyl acetate communication. Overall, findings of the present study showed that NlugCSP10 could selectively interrelate with numerous volatiles emitted from host plants and these ligands could be designated to develop slow‐release mediators that attract/repel N. lugens and subsequently improve the exploration of plans to control this insect pest.     

A comparative study on the heparin‐binding proteomes of Toxoplasma gondii and Plasmodium falciparum

Toxoplasma gondii is an obligatory intracellular apicomplexan parasite which exploits host cell surface components in cell invasion and intracellular parasitization. Sulfated glycans such as heparin and heparan sulfate have been reported to inhibit cell invasion by T. gondii and other apicomplexan parasites such as Plasmodium falciparum. The aim of this study was to investigate the heparin‐binding proteome of T. gondii. The parasite‐derived components were affinity‐purified on the heparin moiety followed by MS fingerprinting of the proteins. The heparin‐binding proteins of T. gondii and P. falciparum were compared based on functionality and affinity to heparin. Among the proteins identified, the invasion‐related parasite ligands derived from tachyzoite/merozoite surface and the secretory organelles were prominent. However, the profiles of the proteins were different in terms of affinity to heparin. In T. gondii, the proteins with highest affinity to heparin were the intracellular components with functions of parasite development contrasted to that of P. falciparum, of which the rhoptry‐derived proteins were prominently identified. The profiling of the heparin‐binding proteins of the two apicomplexan parasites not only explained the mechanism of heparin‐mediated host cell invasion inhibition, but also, to a certain extent, revealed that the action of heparin on the parasite extended after endocytosis.     

Iron inhibits Escherichia coli topoisomerase I activity by targeting the first two zinc‐binding sites in the C‐terminal domain

Escherichia coli DNA topoisomerase I (TopA) contains a 67 kDa N‐terminal catalytic domain and a 30 kDa C‐terminal zinc‐binding region (ZD domain) which has three adjacent tetra‐cysteine zinc‐binding motifs. Previous studies have shown that E. coli TopA can bind both iron and zinc, and that iron binding in TopA results in failure to unwind the negatively supercoiled DNA. Here, we report that each E. coli TopA monomer binds one atom of iron via the first two zinc‐binding motifs in ZD domain and both the first and second zinc‐binding motifs are required for iron binding in TopA. The site‐directed mutagenesis studies further reveal that while the mutation of the third zinc‐binding motif has very little effect on TopA's activity, mutation of the first two zinc‐binding motifs in TopA greatly diminishes the topoisomerase activity in vitro and in vivo, indicating that the first two zinc‐binding motifs in TopA are crucial for its function. The DNA‐binding activity assay and intrinsic tryptophan fluorescence measurements show that iron binding in TopA may decrease the single‐stranded (ss) DNA‐binding activity of ZD domain and also change the protein structure of TopA, which subsequently modulate topoisomerase activity.     

Histone modification and signalling cascade of the dormancy‐associated MADS‐box gene,PpMADS13‐1, in Japanese pear (Pyrus pyrifolia) during endodormancy

Dormancy‐associated MADS‐box (DAM) genes play an important role in endodormancy phase transition. We investigated histone modification in the DAM homolog (PpMADS13‐1) from Japanese pear, via chromatin immunoprecipitation–quantitative PCR, to understand the mechanism behind the reduced expression of the PpMADS13‐1 gene towards endodormancy release. Our results indicated that the reduction in the active histone mark by trimethylation of the histone H3 tail at lysine 4 contributed to the reduction of PpMADS13‐1 expression towards endodormancy release. In contrast, the inactive histone mark by trimethylation of the histone H3 tail at lysine 27 in PpMADS13‐1 locus was quite low, and these levels were more similar to a negative control [normal mouse immunoglobulin G (IgG)] than to a positive control (AGAMOUS) in endodormancy phase transition. The loss of histone variant H2A.Z also coincided with the down‐regulation of PpMADS13‐1. Subsequently, we investigated the PpMADS13‐1 signalling cascade and found that PpCBF2, a pear C‐repeated binding factor, regulated PpMADS13‐1 expression via interaction of PpCBF2 with the 5′‐upstream region of PpMADS13‐1 by transient reporter assay. Furthermore, transient reporter assay confirmed no interaction between the PpMADS13‐1 protein and the pear FLOWERING LOCUS T genes. Taken together, our results enhance understanding of the molecular mechanisms underlying endodormancy phase transition in Japanese pear.     

二化螟Minus-C气味结合蛋白的分子克隆及功能鉴定

气味结合蛋白（odorant binding proteins, OBPs）在昆虫对寄主气味的感受中起重要作用, 但有关Minus-C OBP及其功能的报道很少。本研究通过基因组数据分析并利用RACE技术, 克隆和鉴定了二化螟Chilo suppressalis (Walker)的一个Minus-C OBP基因, 命名为CsupOBP1（GenBank登录号： KC492498）。CsupOBP1基因的开放阅读框长423 bp, 编码141个氨基酸, 其中N端的18个氨基酸为预测的信号肽序列, 成熟蛋白序列中具有4个保守的半胱氨酸位点。实时定量PCR分析显示, 该基因在幼虫头部及成虫雌雄足、 翅和雄性触角等化感组织中高表达, 其中在雄虫触角内的表达量显著高于雌虫触角。利用荧光竞争结合实验对CsupOBP1重组蛋白与38种化合物的结合特性的测定表明, 重组CsupOBP1与β 紫罗兰酮的结合能力最强（Ki=9.53 μmol/L）。触角电位测定表明, 二化螟成虫可对β-紫罗兰酮产生显著的触角电生理反应, 但雄虫反应明显强于雌虫, 与结合试验及雄虫触角中CsupOBP1的表达量显著高于雌虫触角的测定结果相一致。鉴于β-紫罗兰酮是水稻等植物中普遍存在的一种芳香气味组分, 推测CsupOBP1可能通过对该气味的结合和运输, 从而在二化螟对寄主植物的嗅觉定向中起作用。     

DNA‐binding activity and cytotoxic and cell‐cycle arrest properties of some new coumarin derivatives: a multispectral and computational investigation

Coumarins are the most important class of natural compounds found widely in various plants. Many coumarin derivatives with different biological and pharmacological activities have been synthesized. In this study, the antiapoptotic and cytotoxic effects and DNA‐binding properties of some synthetic coumarin derivatives (4b, 4d, 4f, 4 g (DBP‐g), 4 h and 4j) against K562 cell lines were investigated using different techniques. MTT assay indicated that the DBP‐g compound was more active than other derivatives, with a IC₅₀ value of 55 μM, and therefore this compound was chosen for further investigation. Apoptosis induction was assessed using acridine orange/ethidium bromide double‐staining and cell‐cycle analysis. In addition, in vitro DNA‐binding studies were carried out using ultraviolet–visible light absorption and fluorescence spectroscopy, as well as viscosity measurement and molecular modelling studies. In vitro results indicated that DBP‐g interacted with DNA through a groove‐binding mode with a binding constant (K_b) of 1.17 × 10⁴ M^?1. In agreement with other experimental data, molecular docking studies showed that DBP‐g is a minor groove binder. Overall, it can be concluded that DBP‐g could be used as an effective and novel chemotherapeutic agent.     

Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes

Thermodynamic studies on ligand–protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer‐based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4–7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl‐ligand with hexyl spacer. The selectivity in the series of dansyl‐ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH⁰/ΔG⁰. The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design. Copyright © 2014 John Wiley & Sons, Ltd.     

松墨天牛OBP基因MaltOBP2和MaltOBP6的克隆、序列分析及组织表达谱和原核表达研究

【目的】本研究旨在探索松墨天牛Monochamus alternatus Hope在嗅觉识别寄主植物过程中扮演重要角色的气味结合蛋白(odorant binding proteins,OBPs)的结构及功能。【方法】利用生物信息学方法对得到的Malt OBP2和Malt OBP6基因序列和蛋白结构进行分析,并通过实时荧光定量PCR分析Malt OBP2和Malt OBP6在松墨天牛雄虫不同组织和时空中的表达差异,利用p ET32a(+)原核表达载体对Malt OBP2和Malt OBP6进行了诱导蛋白表达。【结果】本研究得到两个松墨天牛气味结合蛋白基因——Malt OBP2(Gen Bank登录号:KP120891)和Malt OBP6(Gen Bank登录号:KP120892),ORF长度分别为402 bp和408 bp,翻译的氨基酸序列均含有4个保守的半胱氨酸位点,表明得到的两个OBP基因的编码蛋白均属于Minus-C OBP亚家族;推导的两个OBP蛋白均有6个α螺旋区域,且α螺旋区域在两个蛋白的位置非常相似,但是两个OBP蛋白推测的配体结合位点和位点极性却完全不同。组织表达模式表明,Malt OBP2和Malt OBP6在成虫头部、触角、下颚(唇)须、腹部末端和足中均有表达,表达程度不一,但都在头部显著表达,触角中的表达量相比其他组织中较低或只是持平。发育表达结果表明,Malt OBP2在蛹触角中的表达量最高,而Malt OBP6在幼虫头部的表达量最高。本研究成功构建了原核表达载体p ET32aMalt OBP2和p ET32a-Malt OBP6,并进行了OBP蛋白诱导表达,低温(16℃和20℃)条件利于蛋白表达在上清液中,延长诱导表达时间(12 h)可以增加蛋白的表达量。【结论】本研究从松墨天牛体内得到了Minus-C OBP蛋白亚家族的两个基因Malt OBP2和Malt OBP6,通过配体结合位点推测它们具有不同的生理功能;通过组织表达谱结果推测这两个OBP基因在松墨天牛中的功能不仅仅局限于嗅觉识别,或还有味觉感受、化学感受等其他生理功能。本研究结果为两个OBP蛋白的结构和功能研究奠定了基础,为探索松墨天牛的化学感受机制提供了条件。