Three Amino Acid Residues Bind Corn Odorants to McinOBP1 in the Polyembryonic Endoparasitoid of Macrocentrus cingulum Brischke

Odorant binding proteins (OBPs) play a central role in transporting odorant molecules from the sensillum lymph to olfactory receptors to initiate behavioral responses. In this study, the OBP of Macrocentrus cingulum McinOBP1 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR experiments indicate that the McinOBP1 is expressed mainly in adult antennae, with expression levels differing by sex. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the McinOBP1 can bind green-leaf volatiles, including aldehydes and terpenoids, but also can bind aliphatic alcohols with good affinity, in the order trans-2-nonenal>cis-3-hexen-1-ol>trans-caryophelle, suggesting a role of McinOBP1 in general odorant chemoreception. We chose those three odorants for further homology modeling and ligand docking based on their binding affinity. The Val58, Leu62 and Glu130 are the key amino acids in the binding pockets that bind with these three odorants. The three mutants, Val58, Leu62 and Glu130, where the valine, leucine and glutamic residues were replaced by alanine, proline and alanine, respectively; showed reduced affinity to these odorants. This information suggests, Val58, Leu62 and Glu130 are involved in the binding of these compounds, possibly through the specific recognition of ligands that forms hydrogen bonds with the ligands functional groups.     

Molecular cloning,expression profile,odorant affinity,and stability of two odorant‐binding proteins in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

The polyembryonic endoparasitoid wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is deployed successfully as a biocontrol agent for corn pest insects from the Lepidopteran genus Ostrinia in Europe and throughout Asia, including Japan, Korea, and China. The odorants are recognized, bound, and solubilized by odorant‐binding protein (OBP) in the initial biochemical recognition steps in olfaction that transport them across the sensillum lymph to initiate behavioral response. In the present study, we examine the odorant‐binding effects on thermal stability of McinOBP2, McinOBP3, and their mutant form that lacks the third disulfide bonds. Real‐time PCR experiments indicate that these two are expressed mainly in adult antennae, with expression levels differing by sex. Odorant‐binding affinities of aldehydes, terpenoids, and aliphatic alcohols were measured with circular dichroism spectroscopy based on changes in the thermal stability of the proteins upon their affinities to odorants. The obtained results reveal higher affinity of trans‐caryophelle, farnesene, and cis‐3‐Hexen‐1‐ol exhibits to both wild and mutant McinOBP2 and McinOBP3. Although conformational flexibility of the mutants and shape of binding cavity make differences in odorant affinity between the wild‐type and mutant, it suggested that lacking the third disulfide bond in mutant proteins may have chance to incorrect folded structures that reduced the affinity to these odorants. In addition, CD spectra clearly indicate proteins enriched with α‐helical content.     

Acceptability and suitability of the European Ostrinia nubilalis Hübner for Macrocentrus cingulum Brischke from Asia and Europe

We examined whether Macrocentrus cingulum (Hymenoptera: Braconidae) of Asian origin could serve as a biological control agent of the maize pest Ostrinia nubilalis (Lepidoptera: Crambidae) in Europe. M. cingulum is already present in Europe, where it does not parasitize O. nubilalis but Ostrinia scapulalis, a related species feeding on wild dicotyledons. In contrast, M. cingulum have been imported from Europe and Asia into North America (where O. nubilalis had been accidentally introduced from Europe), and does parasitize O. nubilalis there. We conducted laboratory infestations to assess host acceptability (parasitoid’s propensity to oviposit) and suitability (parasitoid’s ability to develop) of European O. nubilalis for M. cingulum of European and Asian origin, and of Ostrinia furnacalis (their original host) for Asian M. cingulum. Asian M. cingulum parasitized European O. nubilalis as readily as O. furnacalis, and developed equally well in terms of: % female cocoons, time to first emergence from the cocoon, total number of adult offspring, % female offspring and adult longevity. Adult female parasitoids were significantly larger when emerging from O. nubilalis, mixed-sex and male cocoons were significantly more and less frequent, respectively. The acceptability of O. nubilalis was significantly lower for European than for Asian M. cingulum, and its suitability was zero. Asian M. cingulum appears a potential candidate for introduction as a biological control agent of a major maize pest, European O. nubilalis, provided environmental impact studies, economic analyses, and foreseeable interactions with other biological control agents such as the egg parasitoid Trichogramma brassicae (Hymenoptera: Trichogrammatidae) are satisfying.     

Proteomic analysis of castor bean tick Ixodes ricinus: a focus on chemosensory organs

In arthropods, the large majority of studies on olfaction have been focused on insects, where most of the proteins involved have been identified. In particular, chemosensing in insects relies on two families of membrane receptors, olfactory/gustatory receptors (ORs/GRs) and ionotropic receptors (IRs), and two classes of soluble proteins, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). In other arthropods, such as ticks and mites, only IRs have been identified, while genes encoding for OBPs and CSPs are absent. A third class of soluble proteins, called Niemann-Pick C2 (NPC2) has been suggested as potential carrier for semiochemicals both in insects and other arthropods.Here we report the results of a proteomic analysis on olfactory organs (Haller's organ and palps) and control tissues of the tick Ixodes ricinus, and of immunostaining experiments targeting NPC2s. Adopting different extraction and proteomic approaches, we identified a large number of proteins, and highlighted those differentially expressed. None of the 13 NPC2s known for this species was found. On the other hand, using immunocytochemistry, we detected reaction against one NPC2 in the Haller's organ and palp sensilla. We hypothesized that the low concentration of such proteins in the tick's tissues could possibly explain the discrepant results. In ligand-binding assays the corresponding recombinant NPC2 showed good affinity to the fluorescent probe N-phenylnaphthylamine and to few organic compounds, supporting a putative role of NPC2s as odorant carriers.     

Functional characterization of a Niemann–Pick type C2 protein in the parasitoid wasp Microplitis mediator

Niemann–Pick type C2 (NPC2) is a type of small soluble protein involved in lipid metabolism and triglyceride accumulation in vertebrates and arthropods. Recent studies have determined that NPC2 also participates in chemical communication of arthropods. In this work, two novel NPC2 proteins (MmedNPC2a and MmedNPC2b) in Microplitis mediator were identified. Real‐time quantitative PCR (qPCR) analysis revealed that MmedNPC2a was expressed higher in the antennae than in other tissues of adult wasps compared with MmedNPC2b. Subsequent immunolocalization results demonstrated that NPC2a was located in the lymph cavities of sensilla placodea. To further explore the binding characterization of recombinant MmedNPC2a to 54 candidate odor molecules, a fluorescence binding assay was performed. It was found MmedNPC2a could not bind with selected fatty acids, such as linoleic acid, palmitic acid, stearic acid and octadecenoic acid. However, seven cotton volatiles, 4‐ethylbenzaldehyde, 3,4‐dimethylbenzaldehyde, β‐ionone, linalool, m‐xylene, benzaldehyde and trans‐2‐hexen‐1‐al showed certain binding abilities with MmedNPC2a. Moreover, the predicted 3D model of MmedNPC2a was composed of seven β‐sheets and three pairs of disulfide bridges. In this model, the key binding residues for oleic acid in CjapNPC2 of Camponotus japonicus, Lue68, Lys69, Lys70, Phe97, Thr103 and Phe127, are replaced with Phe85, Ser86, His87, Leu113, Tyr119 and Ile143 in MmedNPC2a, respectively. We proposed that MmedNPC2a in M. mediator may play roles in perception of plant volatiles.     

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (−7.2 to −7.0 kcal/mol) and the cowpea bean dipeptide Lys-Asp (−7.0 kcal/mol) had higher binding affinities than ezetimibe (−6.6 kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (−7.2 kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption.     

Sequence Alignment Reveals Possible MAPK Docking Motifs on HIV Proteins

Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs). MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.     

A conserved domain in arthropod cuticular proteins binds chitin

Many insect cuticular proteins include a 35-36 amino acid motif known as the R&R consensus. The extensive conservation of this region led to the suggestion that it functions to bind chitin. Provocatively, it has no sequence similarity to the well-known cysteine-containing chitin-binding domain found in chitinases and some peritrophic membrane proteins. Using fusion proteins expressed in E. coli, we show that an extended form of the R&R consensus from proteins of hard cuticles is necessary and sufficient for chitin binding. Recombinant AGCP2b, a putative cuticular protein from the mosquito Anopheles gambiae, was expressed in E. coli and the purified protein shown to bind to chitin beads. A stretch of 65 amino acids from AGCP2b, including the R&R consensus, conferred chitin binding to glutathione-S-transferase (GST). Directed mutagenesis of some conserved amino acids within this extended R&R consensus from hard cuticle eliminated chitin binding. Thus arthropods have two distinct classes of chitin binding proteins, those with the chitin-binding domain found in lectins, chitinases and peritrophic membranes (cysCBD) and those with the cuticular protein chitin-binding domain (non-cysCBD).     

烟实夜蛾触角普通气味结合蛋白Ⅱ cDNA的克隆、序列分析及在大肠杆菌中的表达

利用RT PCR技术扩增了编码烟实夜蛾Helicoverpa assulta雌、雄虫触角普通气味 结合蛋白Ⅱ的Cdna片段,将其克隆至Pgem-T Easy载体,获得了普通气味结合蛋白Ⅱ基因成熟蛋白阅读框序列。将该基因重组到表达型质粒Pet-30a(+)中,并转化入原核细胞中表达。序列 测定结果表明,烟实夜蛾触角普通气味结合蛋白基因的成熟蛋白阅读框全长489 bp,编码162个 氨基酸残基,预测分子量和等电点分别为18.2 kD和5.35。推导的氨基酸序列与已报道的10种昆虫普通气味结合蛋白Ⅱ高度同源（73%～98%）,并具有气味结合蛋白的典型特征。SDS-PAGE和Western印迹分析表明,经IPTG诱导,普通气味结合蛋白Ⅱ基因能在大肠杆菌BL21(DE3)中表达,电泳检测到一条约23 kD大小的外源蛋白,与预测的融合蛋白分子量大小相应。     

Molecular dynamics simulations reveal structural differences among wild-type NPC1 protein and its mutant forms

Abstract

NPC1 is a 25-exon gene located on the long arm of chromosome 18q11.2 and encodes NPC1, a transmembrane protein comprising 1278 amino acid residues. Mutations in the NPC1 gene can cause Niemann-Pick disease type C (NP-C), a rare autosomal-recessive neurovisceral disease. We assessed mutant protein folding using computer-based molecular dynamics (MD) simulations and molecular docking of the three most common NPC1 mutations, all of which result in changes in a cysteine-rich luminal loop region of the protein: a) I1061T is the most commonly detected variant in patients with NP-C worldwide; b) P1007A is the second most common variant, frequently detected in Portuguese, British and German patients; c) G992W occurs most often in patients of Acadian descent. Analyses of molecular structural information and related cellular physiological processes revealed that mutant NPC1 proteins exhibited altered function despite being far from the N-terminal domain cholesterol binding. MD simulations revealed that mutant I1061T protein shows remarkable instability in comparison the WT and also de other mutants, and interestingly this mutant has been identified as the most common variant. In the case of the mutant P1007A, it is presumed that this substitution promotes larger structural changes than proline due to their greater hydrophobic properties.

Structural changes related to the G992W mutation may affect the physicochemical space of G992W variant protein because tryptophan induces hydrophobic interactions. Cholesterol docking studies focused on binding recognition showed differences in the binding positions of variants versus the wild-type protein that go some way to explaining the molecular pathogenesis.

Communicated by Ramaswamy H. Sarma     

Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation

Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, β-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or R_p-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N⁶-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.     

In silico studies on marine actinomycetes as potential inhibitors for Glioblastoma multiforme

Glioblastoma multiforme (GBM) is considered to be the most common and often deadly disorder which affects the brain. It is caused by the over expression of proteins such as ephrin type-A receptor 2 (EphA2), epidermal growth factor receptor (EGFR) and EGFRvIII. These 3 proteins are considered to be the potential therapeutic targets for GBM. Among these, EphA2 is reported to be over-expressed in ˜90% of GBM. Herein we selected 35 compounds from marine actinomycetes, 5 in vitro and in vivo studied drug candidates and 4 commercially available drugs for GBM which were identified from literature and analysed by using comparative docking studies. Based on the glide scores and other in silico parameters available in Schrödinger, two selected marine actinomycetes compounds which include Tetracenomycin D and Chartreusin exhibited better binding energy among all the compounds studied in comparative docking. In this study we have demonstrated the inhibition of the 3 selected targets by the two bioactive compounds from marine actinomycetes through in-silico docking studies. Furthermore molecular dynamics simulation were also been performed to check the stability and the amino acids interacted with the 3 molecular targets (EphA2 receptor, EGFR, EGFRvIII) for GBM. Our results suggest that Tetracinomycin D and Chartreusin are the novel and potential inhibitor for the treatment of GBM.