Identity and expression pattern of chemosensory proteins in Heliothis virescens (Lepidoptera, Noctuidae)

Analyzing the chemosensory organs of the moth Heliothis virescens, three proteins belonging to the family of insect chemosensory proteins (CSPs) have been cloned; they are called HvirCSP1, HvirCSP2 and HvirCSP3. The HvirCSPs show about 50% identity between each other and 30–76% identity to CSPs from other species. Overall, they are rather hydrophilic proteins but include a conserved hydrophobic motif. Tissue distribution and temporal expression pattern during the last pupal stages were assessed by Northern blots. HvirCSP mRNAs were detected in various parts of the adult body with a particular high expression level in legs. The expression of HvirCSP1 in legs started early during adult development, in parallel with the appearance of the cuticle. HvirCSP1 mRNA was detectable five days before eclosion (day E-5), increased dramatically on day E-3 and remained at high level into adult life. The tissue distribution and the time course of appearance of HvirCSPs are in agreement with a possible role in contact chemosensation.     

Identity and expression pattern of chemosensory proteins in Heliothis virescens (Lepidoptera, Noctuidae)

Analyzing the chemosensory organs of the moth Heliothis virescens, three proteins belonging to the family of insect chemosensory proteins (CSPs) have been cloned; they are called HvirCSP1, HvirCSP2 and HvirCSP3. The HvirCSPs show about 50% identity between each other and 30–76% identity to CSPs from other species. Overall, they are rather hydrophilic proteins but include a conserved hydrophobic motif. Tissue distribution and temporal expression pattern during the last pupal stages were assessed by Northern blots. HvirCSP mRNAs were detected in various parts of the adult body with a particular high expression level in legs. The expression of HvirCSP1 in legs started early during adult development, in parallel with the appearance of the cuticle. HvirCSP1 mRNA was detectable five days before eclosion (day E-5), increased dramatically on day E-3 and remained at high level into adult life. The tissue distribution and the time course of appearance of HvirCSPs are in agreement with a possible role in contact chemosensation.     

Crystal structure of Apis mellifera OBP14, a C-minus odorant-binding protein, and its complexes with odorant molecules

Apis mellifera (Amel) relies on its olfactory system to detect and identify new-sources of floral food. The Odorant-Binding Proteins (OBPs) are the first proteins involved in odorant recognition and interaction, before activation of the olfactory receptors. The Amel genome possess a set of 21 OBPs, much fewer compared to the 60-70 OBPs found in Diptera genomes. We have undertaken a structural proteomics study of Amel OBPs, alone or in complex with odorant or model compounds. We report here the first 3D structure of a member of the C-minus class OBPs, AmelOBP14, characterized by only two disulfide bridges of the three typical of classical OBPs. We show that AmelOBP14 possesses a core of 6 α-helices comparable to that of classical OBPs, and an extra exposed C-terminal helix. Its binding site is located within this core and is completely closed. Fluorescent experiments using 1-NPN displacement demonstrate that AmelOBP14 is able to bind several compounds with sub micromolar dissociation constants, among which citralva and eugenol exhibit the highest affinities. We have determined the structures of AmelOBP14 in complex with 1-NPN, eugenol and citralva, explaining their strong binding. Finally, by introducing a double cysteine mutant at positions 44 and 97, we show that a third disulfide bridge was formed in the same position as in classical OBPs without disturbing the fold of AmelOBP14.     

Identification and genomic structure of chemosensory proteins (CSP) and odorant binding proteins (OBP) genes expressed in foreleg tarsi of the swallowtail butterfly Papilio xuthus

Chemoreception is a key feature for selection of host plants by phytophagous insects. Female swallowtail butterflies recognize their host plants using chemosensilla present on foreleg tarsi. We constructed a cDNA library of female tarsi and a genome library of Papilio xuthus. We identified 11 chemosensory protein (CSP) genes and three odorant binding proteins (OBP) genes from the cDNA library and eight additional CSP genes from the genome library using the ESTs as probes. A sequence similarity tree of insect CSPs showed that lepidopteran CSPs constructed big branches of the order. Small numbers of CSPs have been identified from the whole genomes of several insect orders which belong to branches separated from those of Lepidoptera. The CSP gene family of Lepidoptera may have diverged in at least two steps, the first on a small scale and the second on a large scale before and after the diversification of insect orders, respectively. Seventeen of 19 CSP genes of P. xuthus clustered in a specific region of the genome, suggesting that they were diversified by gene duplication from a common ancestral gene.     

FZ6基因及其蛋白的生物信息学分析

目的:对frizzled6(FZ6)基因和Frizzled6(Fz6)蛋白进行生物信息学分析,更多的了解该基因的相关信息,为进一步研究Wnt-Fz信号通路以及FZ基因与神经管发育缺陷相关性研究提供基础。方法:运用Internet上的数据库及程序,对FZ6基因结构、单核苷酸多态性(single nucleotide polymorphisms,SNPs)位点、Fz6蛋白的二级结构、蛋白相互作用网络以及Fz蛋白家族的多重序列比对进行生物信息学分析。结果:FZ6基因染色体定位于8q22.3-q23.1,基因全长33995bp,编码706个氨基酸;编码区存在8个SNPs位点,其中错义SNPs4个;多序列比对结果显示10个Fz蛋白家族成员分为4个亚类,Fz3和Fz6为同一类;Fz6蛋白有2个保守的结构域,蛋白序列羧基端缺少其他Fz蛋白普遍存在的S/T-X-V序列模式;其二级结构以α螺旋和随机卷曲为主。结论:通过对FZ6基因及其蛋白的生物信息学分析获得了其相应的生物学特征,为进一步研究奠定基础。     

家蚕化学感受蛋白CSP16的表达及结合特性分析

【目的】化学感受蛋白(chemosensory proteins,CSPs)在昆虫化学信号的感受识别和生长发育调节等生理过程具有重要作用。本研究旨在探索CSP16在家蚕Bombyx mori中的功能。【方法】利用RT-q PCR分析csp16在家蚕不同发育时期和不同组织的表达特征,用原核表达系统对家蚕CSP16进行表达纯化,并通过荧光结合实验检测该蛋白与不同配体化合物的结合特性。【结果】RT-q PCR结果表明,csp16在家蚕1-5龄幼虫中呈规律性表达,各龄期眠蚕中表达量最高,5龄第3天幼虫中主要表达于头、表皮、精巢和卵巢等。蜕皮激素(20E)处理后csp16在不同龄期幼虫和5龄幼虫不同组织中的表达量上调。纯化后CSP16的荧光结合实验表明,CSP16与醇类、酯类、醛类、酚类和苯环类等化合物的亲和力都较弱。【结论】csp16在家蚕不同龄期幼虫眠蚕中表达量最高,蜕皮激素使csp16在家蚕取食幼虫中的表达量出现上调,提示其可能参与家蚕幼虫的蜕皮过程。     

Effects of androgens on behavioral and vomeronasal responses to chemosensory cues in male terrestrial salamanders (Plethodon shermani)

Chemosensory stimuli and sex steroid hormones are both required for the full expression of social behaviors in many species. The terrestrial salamander, Plethodon shermani, is an emerging nonmammalian system for investigating the nature and evolution of pheromonal communication, yet little is known regarding the role of sex steroid hormones. We hypothesized that increased circulating androgen levels in male P. shermani enhance chemoreception through morphological, behavioral, and physiological mechanisms. Experimental elevation of plasma androgens increased development of cirri, morphological structures thought to enhance the transfer of chemosensory cues from the substrate to the vomeronasal organ (VNO). Elevated plasma androgens also increased expression of a chemo-investigatory behavior (nose tapping) and increased preference for some female-derived chemosensory cues. Male-produced courtship pheromones activated a large number of cells in the VNO as measured by the method of agmatine uptake. However, androgen levels did not affect the total number of vomeronasal cells activated by male-produced courtship pheromones. Future studies will determine whether androgens potentially modulate responsiveness of the VNO to female-derived (as opposed to male-derived) chemosensory cues.     

中华蜜蜂信息素结合蛋白OBP10的基因克隆、原核表达和配基结合特性分析

【目的】气味结合蛋白在中华蜜蜂Apis cerana cerana嗅觉系统中起到重要作用,本实验拟研究中华蜜蜂一个新的信息素结合蛋白OBP10及其与蜜蜂信息素以及蜜源开花植物挥发物的结合特性。【方法】本实验通过RT-PCR扩增获得OBP10基因全长(Gen Bank登录号:KP717060.1),以p ET-30a构建原核表达载体,并以Ni2+琼脂糖柱进行重组蛋白表达和分离纯化,在N-苯基-1-萘胺(N-phenyl-1-naphthylamine,1-NPN)作为荧光报告子下利用荧光光谱法体外研究重组Acer OBP10与多种候选化学配基的结合特征。【结果】经多序列联配分析,发现Acer OBP10的多个同源基因均为信息素结合蛋白(pheromone binding proteins,PBPs)。配基结合特性分析显示,Acer OBP10对14种候选配基中的蜂王信息素成分对羟基苯甲酸甲酯(HOB)竞争力最大,相对荧光可降至6.06%,解离常数11.04μmol/L;进一步表明Acer OBP10属于一个新的中蜂PBPs。此外,Acer OBP10也能和包括工蜂信息素(香叶醇和橙花醇)、报警信息素(2-庚酮和乙酸异戊酯)等蜜蜂信息素以及蜜源开花植物挥发物之一的β-紫罗兰酮结合,表明Acer OBP10可能是一种以信息素结合为主的多功能结合蛋白。【结论】Acer OBP10是中蜂一个新的信息素结合蛋白,与此前我们鉴定的蜂王信息素结合蛋白Acer ASP1相比,Acer OBP10对蜜蜂信息素的结合谱更为广泛,这些结果将对进一步研究中华蜜蜂信息素识别和传递提供理论基础,对于深入了解中华蜜蜂嗅觉影响生理功能的行为机制具有重要意义。     

Role of adipocyte lipid-binding protein (ALBP) and acyl-CoA binding protein (ACBP) in PPAR-mediated transactivation

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by a number of fatty acids and fatty acid derivatives. By contrast, we have recently shown that acyl-CoA esters display PPAR antagonistic properties in vitro. We have also shown that the adipocyte lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization. We therefore speculated that FABPs and ACBP might regulate the availability of PPAR agonists and antagonists by affecting not only their esterification in the cytoplasm but also their transport to and availability in the nucleus. We show here that coexpression of ALBP or ACBP exerts a negative effect on ligand-dependent PPAR transactivation, when tetradecylthioacetic (TTA) is used as ligand but not when the thiazolidinedione BRL49653 is used as ligand. The results presented here do not support the hypothesis that ALBP facilitates the transport of the fatty acid-type ligands to the nucleus, rather ALBP appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands.     

Oxygen-evolving extrinsic proteins (PsbO,P,Q,R): Bioinformatic and functional analysis

The water-splitting and oxygen-evolving (OE) reaction is carried out by a large multisubunit protein complex, Photosystem II (PSII), that has two distinct regions: a membrane intrinsic-region that includes most of the PSII subunits and a lumenal extrinsic-region that is in close association to the manganese catalytic center. The recently determined PSII 3D structures from cyanobacteria provide a considerable amount of new knowledge about the OE architecture (K.N. Ferreira, T.M. Iverson, K. Maghlaoui, J. Barber, S. Iwata, Architecture of the photosynthetic oxygen-evolving center, Science 303 (2004) 1831-1838; B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II, Nature 438 (2005) 1040-1044). Most of the intrinsic core PSII polypeptides have been well conserved through evolution from ancient cyanobacteria to modern plants, keeping the essence of PSII light driven reactions from prokaryotes to eukaryotes; but what is striking is the large number of changes that have occurred in the oxygen-evolving extrinsic proteins (OEEp) associated to PSII lumenal side. For unknown reasons plant PSII has required the “invention” of three OEEps: PsbP (23 kDa), PsbQ (16 kDa) and PsbR (10 kDa); associated to the ubiquitous OEEp PsbO (33 kDa). This set of proteins seems to be required in plants for the full activity and stability of the OE center in vivo, but their specific function is not clear. In this paper, bioinformatics and functional data show that the OEEps present in plants and green algae are very distinct from their prokaryotic counterparts. Moreover, clear differences are found for PsbQ from higher plants and green algae; and a relationship has been found between PsbR and the Mn cluster.     

Stability,protein binding and thiol interaction studies on [2-acetoxy-(2-propynyl)benzoate]hexacarbonyldicobalt

Cobalt–alkyne complexes are a new class of potent cytotoxic drugs. The lead compound [2-acetoxy-(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS) showed high effects on several human cancer cell lines. In order to evaluate further the in-vitro properties and reactivity of this substance we performed stability and protein binding studies and investigated the interaction of this complex with 1,2-ethanedithiol, L-cysteine and glutathione. UV–Vis, HPLC and AAS studies showed that the compound was sufficiently stable under in-vitro conditions. Binding to human serum albumin increased from approximately 25% at the beginning to over 50% after 48 h of incubation as determined by ethanol preciptation and size exclusion chromatography. The interaction with thiols resulted in disulfide bond formation of the thiols.Published online: March 2005     

Receptor-transporting protein 1 short (RTP1S) mediates translocation and activation of odorant receptors by acting through multiple steps

Odorant receptor (OR) proteins are retained in the endoplasmic reticulum when heterologously expressed in cultured cells of non-olfactory origins. RTP1S is an accessory protein to mammalian ORs and facilitates their trafficking to the cell-surface membrane and ligand-induced responses in heterologous cells. The mechanism by which RTP1S promotes the functional expression of ORs remains poorly understood. To obtain a better understanding of the role(s) of RTP1S, we performed a series of structure-function analyses of RTP1S in HEK293T cells. By constructing RTP1S deletion and chimera series and subsequently introducing single-site mutations into the protein, we found the N terminus of RTP1S is important for the endoplasmic reticulum exit of ORs and that a middle region of RTP1S is important for OR trafficking from the Golgi to the membrane. Using sucrose gradient centrifugation, we found that the localization of RTP1S to the lipid raft microdomain is critical to the activation of ORs. Finally, in a protein-protein interaction analysis, we determined that the C terminus of RTP1S may be interacting with ORs. These findings provide new insights into the distinct roles of RTP1S in OR translocation and activation.     

重金属结合肽（蛋白）的结构分析

金属离子与金属结合肽（蛋白）的相互作用与应用研究,一直是生物无机化学的重点和热点,也是分子间相互作用研究领域的难点。本研究利用ClustalX、BLAST等生物信息技术与方法对大量已知的重金属结合肽进行分析与数据挖掘。确定筛选获得的重金属结合肽常富含His,无Cys,无金属结合肽模式序列,进化不保守;部分氨基酸序列结构（如六肽）可在蛋白数据库中找到相似序列。序列特征主要为Zn^2＋相关的转录因子。本研究为重金属结合蛋白-重金属离子的相互作用分析简化为重金属结合肽-重金属离子的结构模拟与分析提供了重要的理论基础和研究手段。     

Complete mitochondrial genome of the hemp borer,Grapholita delineana (Lepidoptera: Tortricidae): Gene variability and phylogeny among Grapholita

The mitochondrial genome (mitogenome) has been extensively used in phylogenetics and species-level evolutionary investigations. The lepidopteran family Tortricidae (leaf-roller moths), including the genus Grapholita, contains numerous species of economic importance, but for the majority of Grapholita species, their mitogenomes remain poorly studied. Here, we sequence and annotate the full mitogenome of Grapholita delineana, an important pest of hemp worldwide and compare it with the mitogenomes of two congeneric species available from GenBank. The G. delineana mitogenome is 15,599 bp long, including 37 typical mitochondrial genes and an A + T-rich region. Gene content, order and orientation are identical to other reported tortricid mitogenomes. Analyses of nucleotide diversity, Ka/Ks, genetic distance and number of variable sites together suggest that nad6 is the fastest-evolving gene among the mitochondrial PCGs of Grapholita. Our analyses indicate that Grapholita, as presently defined, is not monophyletic, confirming previous morphological and multiple-gene studies, using mitogenomic evidence. Our study provides information on comparative mitogenomics of Tortricidae especially Grapholita.