Bacillus thuringiensis toxin,Cry1C interacts with 128HLHFHLP134 region of aminopeptidase N of agricultural pest,Spodoptera litura

We modeled Cry1C toxin and its Aminopeptidase-N receptor and in silico docking analysis was performed. Further, we utilized biopanning against Cry1C followed by blocking assays and mutagenesis analysis to identify the binding epitope of SlAPN. We have identified a putative SlAPN binding region, APN-CRY (128HLHFHLP134). A derivative of SlAPN carrying the 128HLHFHLP134 region termed as binding region of APN (BR-APN) was cloned and its involvement in Cry1C binding and toxicity was checked. Cry1C-BR-APN binding was competed by synthetic peptides homologous to loop2 and loop3 of domain II but not by that of loopα. Additionally, alanines substitution of residues H128, H130, H132 and P134 affect the binding efficiency of receptor to Cry1C toxin (upto 4-fold lower affinity).These residues are also implicated in Cry1C toxicity as shown by the reduced ability to affect the mortality of Cry1C on S. litura larvae when toxin was preincubated with a fragment of the receptor.     

β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae)

Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [³H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-³H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus.     

Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2

Arylalkylamine N-acyltransferase like 2 (AANATL2) catalyzes the formation of N-acylarylalkylamides from the corresponding acyl-CoA and arylalkylamine. The N-acylation of biogenic amines in Drosophila melanogaster is a critical step for the inactivation of neurotransmitters, cuticle sclerotization, and melatonin biosynthesis. In addition, D. melanogaster has been used as a model system to evaluate the biosynthesis of fatty acid amides: a family of potent cell signaling lipids. We have previously showed that AANATL2 catalyzes the formation of N-acylarylakylamides, including long-chain N-acylserotonins and N-acyldopamines. Herein, we define the kinetic mechanism for AANATL2 as an ordered sequential mechanism with acetyl-CoA binding first followed by tyramine to generate the ternary complex prior to catalysis. Bell shaped k_{cat,app – acetyl-CoA} and (k_cat/K_m)_{app – acetyl-CoA} pH-rate profiles identified two apparent pK_a,app values of ∼7.4 and ∼8.9 that are critical to catalysis, suggesting the AANATL2-catalyzed formation of N-acetyltyramine occurs through an acid/base chemical mechanism. Site-directed mutagenesis of a conserved glutamate that corresponds to the catalytic base for other D. melanogaster AANATL enzymes did not produce a substantial depression in the k_cat,app value nor did it abolish the pK_a,app value attributed to the general base in catalysis (pK_a ∼7.4). These data suggest that AANATL2 catalyzes the formation of N-acylarylalkylamides using either different catalytic residues or a different chemical mechanism relative to other D. melanogaster AANATL enzymes. In addition, we constructed other site-directed mutants of AANATL2 to help define the role of targeted amino acids in substrate binding and/or enzyme catalysis.     

Analyses of α-amylase and α-glucosidase in the malaria vector mosquito,Anopheles gambiae,as receptors of Cry11Ba toxin of Bacillus thuringiensis subsp. jegathesan

Bacillus thuringiensis subsp. jegathesan produces Cry11Ba crystal protein with high toxicity to mosquito larvae. The Cry11Ba toxicity is dependent on its receptors on mosquito larval midgut epithelial cells. Previously, a cadherin-like protein (AgCad2), aminopeptidase (AgAPN2) and alkaline phosphatase (AgALP1) were reported to be involved in regulation of Cry11Ba toxicity on Anopheles gambiae larvae. Here, the cDNAs encoding α-amylase (AgAmy1) and α-glucosidase (Agm3) were cloned from A. gambiae larva midgut. Both are glycophosphatidylinositol (GPI) anchored proteins on brush border membranes (BBMV). Immunohistochemistry revealed their localization on different regions of the larval midgut. AgAmy1 and Agm3 bound Cry11Ba with high affinity, 37.6 nM and 21.1 nM respectively. Cry11Ba toxicity against A. gambiae larvae was neutralized by both AgAmy1 and Agm3. The results provide evidence that both AgAmy1 and Agm3 function as receptors of Cry11Ba in A. gambiae.     

Herbivory-induced glucose transporter gene expression in the brown planthopper,Nilaparvata lugens

Nilaparvata lugens, the brown planthopper (BPH) feeds on rice phloem sap, containing high amounts of sucrose as a carbon source. Nutrients such as sugars in the digestive tract are incorporated into the body cavity via transporters with substrate selectivity. Eighteen sugar transporter genes of BPH (Nlst) were reported and three transporters have been functionally characterized. However, individual characteristics of NlST members associated with sugar transport remain poorly understood. Comparative gene expression analyses using oligo-microarray and quantitative RT-PCR revealed that the sugar transporter gene Nlst16 was markedly up-regulated during BPH feeding. Expression of Nlst16 was induced 2 h after BPH feeding on rice plants. Nlst16, mainly expressed in the midgut, appears to be involved in carbohydrate incorporation from the gut cavity into the hemolymph. Nlst1 (NlHT1), the most highly expressed sugar transporter gene in the midgut was not up-regulated during BPH feeding. The biochemical function of NlST16 was shown as facilitative glucose transport along gradients. Glucose uptake activity by NlST16 was higher than that of NlST1 in the Xenopus oocyte expression system. At least two NlST members are responsible for glucose uptake in the BPH midgut, suggesting that the midgut of BPH is equipped with various types of transporters having diversified manner for sugar uptake.     

The steroid hormone 20-hydroxyecdysone promotes switching from autophagy to apoptosis by increasing intracellular calcium levels

Autophagy regulates cell survival (or cell death in several cases), whereas apoptosis regulates cell death. However, the relationship between autophagy and apoptosis and the regulative mechanism is unclear. We report that steroid hormone 20-hydroxyecdysone (20E) promotes switching from autophagy to apoptosis by increasing intracellular calcium levels in the midgut of the lepidopteran insect Helicoverpa armigera. Autophagy and apoptosis sequentially occurred during midgut programmed cell death under 20E regulation, in which lower concentrations of 20E induced microtubule-associated protein 1 light chain 3–phosphatidylethanolamine (LC3–II, also known as autophagy-related gene 8, ATG8) expression and autophagy. High concentrations of 20E induced cleavage of ATG5 to NtATG5 and pro-caspase-3 to active caspase-3, which led to a switch from autophagy to apoptosis. Blocking autophagy by knockdown of ATG5, ATG7, or ATG12, or with the autophagy inhibitor 3-methyladenine, inhibited 20E-induced autophagy and apoptosis. Blocking apoptosis by using the apoptosis inhibitor Ac-DEVD-CHO did not prevent 20E-induced autophagy, suggesting that apoptosis relies on autophagy. ATG5 knockdown resulted in abnormal pupation and delayed pupation time. High concentrations of 20E induced high levels of intracellular Ca²⁺, NtATG5, and active caspase-3, which mediated the switch from autophagy to apoptosis. Blocking 20E-mediated increase of cellular Ca²⁺ caused a decrease of NtATG5 and active caspase-3 and repressed the transformation from autophagy to apoptosis, thereby promoting cell survival. 20E induces an increase in the concentration of intracellular Ca²⁺, thereby switching autophagic cell survival to apoptotic cell death.     

Enhancement of hydrolytic activity of thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 through optimization of amino acid residues surrounding the substrate binding site

The hydrolytic activity of a thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 (AmyL) toward soluble starch was enhanced through optimization of amino acid (aa) residues situated near the substrate binding site. Twenty-four selected aa residues were replaced with Ala, and Gly429 and Gly550 were altered to Lys and Glu, respectively, based on comparison of AmyL's aa sequence with related enzymes. Y426A, H431A, I509A, and K549A showed notably higher activity than the wild type at 162–254% of wild-type activity. Tyr426, His431, and Ile509 were predicted to be located near subsite −2, while Lys549 was near subsite +2. Ser, Ala, Ala, and Met were found to be the best aa residues for the positions of Tyr426, His431, Ile509, and Lys549, respectively. Combinations of the optimized single mutations at distant positions were effective in enhancing catalytic activity. The double-mutant enzymes Y426S/K549M, H431A/K549M, and I509A/K549M, combining two of the selected single mutations, showed 340%, 252%, and 271% of wild type activity, respectively. Triple and quadruple-mutant enzymes of the selected mutations did not show higher activity than the best double-mutant, Y426S/K549M.     

Identification of critical regions within the TIR domain of IL-1 receptor type I

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC′ helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.     

An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation

GABA-activated RDL receptors are the insect equivalent of mammalian GABA_A receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6′M) ablates picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC₅₀ of WT receptors, enhancing responses, but in T6′M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC₅₀, the picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6′ residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents.     

Positive selection of digestive Cys proteases in herbivorous Coleoptera

Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within –or close to– amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins.     

Structural features,evolutionary relationships,and transcriptional regulation of C-type lectin-domain proteins in Manduca sexta

C-type lectins (CTLs) are a large family of Ca²⁺-dependent carbohydrate-binding proteins recognizing various glycoconjugates and functioning primarily in immunity and cell adhesion. We have identified 34 CTLDP (for CTL-domain protein) genes in the Manduca sexta genome, which encode proteins with one to three CTL domains. CTL-S1 through S9 (S for simple) have one or three CTL domains; immulectin-1 through 19 have two CTL domains; CTL-X1 through X6 (X for complex) have one or two CTL domains along with other structural modules. Nine simple CTLs and seventeen immulectins have a signal peptide and are likely extracellular. Five complex CTLs have both an N-terminal signal peptide and a C-terminal transmembrane region, indicating that they are membrane anchored. Immulectins exist broadly in Lepidoptera and lineage-specific gene duplications have generated three clusters of fourteen genes in the M. sexta genome, thirteen of which have similar expression patterns. In contrast to the family expansion, CTL-S1∼S6, S8, and X1∼X6 have 1:1 orthologs in at least four lepidopteran/dipteran/coleopteran species, suggestive of conserved functions in a wide range of holometabolous insects. Structural modeling suggests the key residues for Ca²⁺-dependent or independent binding of certain carbohydrates by CTL domains. Promoter analysis identified putative κB motifs in eighteen of the CTL genes, which did not have a strong correlation with immune inducibility in the mRNA or protein levels. Together, the gene identification, sequence comparisons, structure modeling, phylogenetic analysis, and expression profiling establish a solid foundation for future studies of M. sexta CTL-domain proteins.     

EWGWS insert in Plasmodium falciparum ookinete surface enolase is involved in binding of PWWP containing peptides: Implications to mosquito midgut invasion by the parasite

There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host–pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids (¹⁰⁴EWGWS¹⁰⁸) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking ¹⁰⁴EWGWS¹⁰⁸, (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed.     

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b₅ enhanced the k_cat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min⁻¹) with little effect on K_M (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species.     

Midgut aminopeptidase N isoforms from Ostrinia nubilalis: Activity characterization and differential binding to Cry1Ab and Cry1Fa proteins from Bacillus thuringiensis

Aminopeptidase N (APN) isoforms from Lepidoptera are known for their involvement in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis. These enzymes belong to a protein family with at least eight different members that are expressed simultaneously in the midgut of lepidopteran larvae. Here, we focus on the characterization of the APNs from Ostrinia nubilalis (OnAPNs) to identify potential Cry receptors. We expressed OnAPNs in insect cells using a baculovirus system and analyzed their enzymatic activity by probing substrate specificity and inhibitor susceptibility. The interaction with Cry1Ab and Cry1Fa proteins (both found in transgenic insect-resistant maize) was evaluated by ligand blot assays and immunocytochemistry. Ligand blots of brush border membrane proteins showed that both Cry proteins bound mainly to a 150 kDa-band, in which OnAPNs were greatly represented. Binding analysis of Cry proteins to the cell-expressed OnAPNs showed that OnAPN1 interacted with both Cry1Ab and Cry1Fa, whereas OnAPN3a and OnAPN8 only bound to Cry1Fa. Two isoforms, OnAPN2 and OnAPN3b, did not interact with any of these two proteins. This work provides the first evidence of a differential role of OnAPN isoforms in the mode of action of Cry proteins in O. nubilalis.     

The substrate binding interface of alkylpurine DNA glycosylase AlkD

Tandem helical repeats have emerged as an important DNA binding architecture. DNA glycosylase AlkD, which excises N3- and N7-alkylated nucleobases, uses repeating helical motifs to bind duplex DNA and to selectively pause at non-Watson–Crick base pairs. Remodeling of the DNA backbone promotes nucleotide flipping of the lesion and the complementary base into the solvent and toward the protein surface, respectively. The important features of this new DNA binding architecture that allow AlkD to distinguish between damaged and normal DNA without contacting the lesion are poorly understood. Here, we show through extensive mutational analysis that DNA binding and N3-methyladenine (3mA) and N7-methylguanine (7mG) excision are dependent upon each residue lining the DNA binding interface. Disrupting electrostatic or hydrophobic interactions with the DNA backbone substantially reduced binding affinity and catalytic activity. These results demonstrate that residues seemingly only involved in general DNA binding are important for catalytic activity and imply that base excision is driven by binding energy provided by the entire substrate interface of this novel DNA binding architecture.     

Differential interactions of 5-(4-piperidyl)-3-isoxazolol analogues with insect γ-aminobutyric acid receptors leading to functional selectivity

γ-Aminobutyric acid (GABA) receptors (GABARs) mediate fast inhibitory synaptic transmission and are also targets for drugs and insecticides. To better understand the molecular interactions of ligands with the orthosteric sites of GABARs, we examined 4-aryl/arylalkyl-5-(4-piperidyl)-3-isoxazolol, 4-aryl-5-(4-piperidyl)-3-isothiazolol, and 5-aryl-4-(4-piperidyl)-1-hydroxypyrazole for their antagonism with regard to three insect GABARs. The 3-isoxazolol was preferable to the 3-isothiazolol and 1-hydroxypyrazole in antagonism to common cutworm and housefly GABARs. Of the tested analogues, 4-(3-biphenylyl)-5-(4-piperidyl)-3-isoxazolol (2a) displayed the greatest antagonism for common cutworm and housefly GABARs, with IC₅₀ values of 3.4 and 10.2 μM, respectively. In contrast to the antagonism of the two GABARs, 2a showed partial agonism for the case of small brown planthopper GABARs, with an EC₅₀ value of 31.3 μM. Homology models and docking simulations revealed that a cation-π interaction between an analogue and an Arg residue in loop C or E of the orthosteric site is a key component of antagonism. This specific phenomenon was lacking in the interactions between 2a and the orthosteric site of small brown planthopper GABARs. These findings provide important insights into designing and developing novel drugs and insecticides.