Wataru Tsuchiya

Niemann–Pick type C2 protein mediating chemical communication in the worker ant

Significance Worker ants are responsible for various tasks for their colony. In their chemical communication, odorant-binding proteins and chemosensory proteins, which accumulate in the sensillum lymph in the antennae, play roles in transporting semiochemicals to chemosensory receptors. However, the number of these proteins is not sufficient to interact with a large number of semiochemicals. Niemann–Pick type C2 protein was identified from the antenna of the worker Japanese carpenter ant, Camponotus japonicus (CjapNPC2). CjapNPC2 accumulated in the sensillum cavity in the basiconic sensillum. The ligand-binding pocket was composed of a flexible β-structure, which allowed binding to various potential semiochemicals, some of which elicited antennal electrophysiological responses. CjapNPC2 might play crucial roles in chemical communication required to perform worker ant tasks. Ants are eusocial insects that are found in most regions of the world. Within its caste, worker ants are responsible for various tasks that are required for colony maintenance. In their chemical communication, α-helical carrier proteins, odorant-binding proteins, and chemosensory proteins, which accumulate in the sensillum lymph in the antennae, play essential roles in transferring hydrophobic semiochemicals to chemosensory receptors. It has been hypothesized that semiochemicals are recognized by α-helical carrier proteins. The number of these proteins, however, is not sufficient to interact with a large number of semiochemicals estimated from chemosensory receptor genes. Here we shed light on this conundrum by identifying a Niemann–Pick type C2 (NPC2) protein from the antenna of the worker Japanese carpenter ant, Camponotus japonicus (CjapNPC2). CjapNPC2 accumulated in the sensillum cavity in the basiconic sensillum. The ligand-binding pocket of CjapNPC2 was composed of a flexible β-structure that allowed it to bind to a wide range of potential semiochemicals. Some of the semiochemicals elicited electrophysiolgical responses in the worker antenna. In vertebrates, NPC2 acts as an essential carrier protein for cholesterol from late endosomes and lysosomes to other cellular organelles. However, the ants have evolved an NPC2 with a malleable ligand-binding pocket as a moderately selective carrier protein in the sensillum cavity of the basiconic sensillum. CjapNPC2 might be able to deliver various hydrophobic semiochemicals to chemosensory receptor neurons and plays crucial roles in chemical communication required to perform the worker ant tasks.

Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori

Juvenile hormone (JH) plays crucial roles in many aspects of the insect life. All the JH actions are initiated by transport of JH in the hemolymph as a complex with JH-binding protein (JHBP) to target tissues. Here, we report structural mechanism of JH delivery by JHBP based upon the crystal and solution structures of apo and JH-bound JHBP. In solution, apo-JHBP exists in equilibrium of multiple conformations with different orientations of the gate helix for the hormone-binding pocket ranging from closed to open forms. JH-binding to the gate-open form results in the fully closed JHBP-JH complex structure where the bound JH is completely buried inside the protein. JH-bound JHBP opens the gate helix to release the bound hormone likely by sensing the less polar environment at the membrane surface of target cells. This is the first report that provides structural insight into JH signaling.

Lon protease negatively affects GacA protein stability and expression of the Gac/Rsm signal transduction pathway in Pseudomonas protegens

In Pseudomonas protegens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway controls secondary metabolism and suppression of fungal root pathogens via the expression of regulatory small RNAs (sRNAs). Because of its high cost, this pathway needs to be protected from overexpression and to be turned off in response to environmental stress such as the lack of nutrients. However, little is known about its underlying molecular mechanisms. In this study, we demonstrated that Lon protease, a member of the ATP-dependent protease family, negatively regulated the Gac/Rsm cascade. In a lon mutant, the steady-state levels and the stability of the GacA protein were significantly elevated at the end of exponential growth. As a consequence, the expression of the sRNAs RsmY and RsmZ and that of dependent physiological functions such as antibiotic production were significantly enhanced. Biocontrol of Pythium ultimum on cucumber roots required fewer lon mutant cells than wild-type cells. In starved cells, the loss of Lon function prolonged the half-life of the GacA protein. Thus, Lon protease is an important negative regulator of the Gac/Rsm signal transduction pathway in P. protegens.

Crystal structure of silkworm Bombyx mori JHBP in complex with 2-methyl-2,4-pentanediol: plasticity of JH-binding pocket and ligand-induced conformational change of the second cavity in JHBP

Juvenile hormones (JHs) control a diversity of crucial life events in insects. In Lepidoptera which major agricultural pests belong to, JH signaling is critically controlled by a species-specific high-affinity, low molecular weight JH-binding protein (JHBP) in hemolymph, which transports JH from the site of its synthesis to target tissues. Hence, JHBP is expected to be an excellent target for the development of novel specific insect growth regulators (IGRs) and insecticides. A better understanding of the structural biology of JHBP should pave the way for the structure-based drug design of such compounds. Here, we report the crystal structure of the silkworm Bombyx mori JHBP in complex with two molecules of 2-methyl-2,4-pentanediol (MPD), one molecule (MPD1) bound in the JH-binding pocket while the other (MPD2) in a second cavity. Detailed comparison with the apo-JHBP and JHBP-JH II complex structures previously reported by us led to a number of intriguing findings. First, the JH-binding pocket changes its size in a ligand-dependent manner due to flexibility of the gate α1 helix. Second, MPD1 mimics interactions of the epoxide moiety of JH previously observed in the JHBP-JH complex, and MPD can compete with JH in binding to the JH-binding pocket. We also confirmed that methoprene, which has an MPD-like structure, inhibits the complex formation between JHBP and JH while the unepoxydated JH III (methyl farnesoate) does not. These findings may open the door to the development of novel IGRs targeted against JHBP. Third, binding of MPD to the second cavity of JHBP induces significant conformational changes accompanied with a cavity expansion. This finding, together with MPD2-JHBP interaction mechanism identified in the JHBP-MPD complex, should provide important guidance in the search for the natural ligand of the second cavity.

Purification, cDNA cloning and recombinant protein expression of a phloem lectin-like anti-insect defense protein BPLP from the phloem exudate of the wax gourd, Benincasa hispida.

Latex and other exudates in plants contain various proteins that are thought to play important defensive roles against herbivorous insects and pathogens. Herein, the defensive effects of phloem exudates against the Eri silkworm, Samia ricini (Saturniidae, Lepidoptera) in several cucurbitaceous plants were investigated. It was found that phloem exudates are responsible for the defensive activities of cucurbitaceous plants, such as the wax gourd Benincasa hispida and Cucumis melo, especially in B. hispida, whose leaves showed the strongest growth-inhibitory activity of all the cucurbitaceous plants tested. A 35 kDa proteinaceous growth-inhibitory factor against insects designated BPLP (B. hispida Phloem Lectin-like Protein) was next isolated and purified from the B. hispida exudate, using anion exchange and gel filtration chromatography. A very low concentration (70 μg/g) of BPLP significantly inhibited growth of S. ricini larvae. The full-length cDNA (1076 bp) encoding BPLP was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of BPLP had 51% identity with a cucurbitaceous phloem lectin (phloem protein 2, PP2), and showed binding specificity to oligomers of N-acetylglucosamine. Some features of BPLP indicated that it does not have a cysteine residue and it is composed of two repeats of similar sequences, suggesting that BPLP is distinct from PP2. Recombinant BPLP, obtained by expressing the cDNA in Escherichia coli, showed both chitin-binding lectin activity and growth-inhibitory activity against S. ricini larvae. The present study thus provides experimental evidence that phloem exudates of Cucurbitaceae plants, analogous to plant latex, play defensive roles against insect herbivores, especially against chewing insects, and contain defensive substances toxic to them.

PHD finger of the SUMO ligase Siz/PIAS family in rice reveals specific binding for methylated histone H3 at lysine 4 and arginine 2

We determined the three‐dimensional structure of the PHD finger of the rice Siz/PIAS‐type SUMO ligase, OsSiz1, by NMR spectroscopy and investigated binding ability for a variety of methylated histone H3 tails, showing that OsSiz1–PHD primarily recognizes dimethylated Arg2 of the histone H3 and that methylations at Arg2 and Lys4 reveal synergy effect on binding to OsSiz1–PHD. The K4 cage of OsSiz1–PHD for trimethylated Lys4 of H3K4me3 was similar to that of the BPTF–PHD finger, while the R2 pocket for Arg2 was different. It is intriguing that the PHD module of Siz/PIAS plays an important role, with collaboration with the DNA binding domain SAP, in gene regulation through SUMOylation of a variety of effectors associated with the methylated arginine‐riched chromatin domains.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity

BackgroundDihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood.ResultsBy using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds.ConclusionsWe isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Control of enzymatic degradation of biodegradable polymers by treatment with biosurfactants, mannosylerythritol lipids, derived from Pseudozyma spp. yeast strains

Cutinase-like esterase from the yeasts Pseudozyma antarctica (PaE) shows strong degradation activity in an agricultural biodegradable plastic (BP) model of mulch films composed of poly(butylene succinate-co-adipate) (PBSA). P. antarctica is known to abundantly produce a glycolipid biosurfactant, mannosylerythritol lipid (MEL). Here, the effects of MEL on PaE-catalyzed degradation of BPs were investigated. Based on PBSA dispersion solution, the degradation of PBSA particles by PaE was inhibited in the presence of MEL. MEL behavior on BP substrates was monitored by surface plasmon resonance (SPR) using a sensor chip coated with polymer films. The positive SPR signal shift indicated that MEL readily adsorbed and spread onto the surface of a BP film. The amount of BP degradation by PaE was monitored based on the negative SPR signal shift and was decreased 1.7-fold by MEL pretreatment. Furthermore, the shape of PBSA mulch films in PaE-containing solution was maintained with MEL pretreatment, whereas untreated films were almost completely degraded and dissolved. These results suggest that MEL covering the surface of BP film inhibits adsorption of PaE and PaE-catalyzed degradation of BPs. We applied the above results to control the microbial degradation of BP mulch films. MEL pretreatment significantly inhibited BP mulch film degradation by both PaE solution and BP-degradable microorganism. Moreover, the degradation of these films was recovered after removal of the coated MEL by ethanol treatment. These results demonstrate that the biodegradation of BP films can be readily and reversibly controlled by a physical approach using MEL.

Enhancement of Biodegradable Plastic-degrading Enzyme Production from Paraphoma-like Fungus, Strain B47-9

To improve the productivity of Paraphoma-like fungal strain B47-9 for biodegradable plastic (BP)-degrading enzyme (PCLE), the optimal concentration of emulsified poly(butylene succinate-co-adipate) (PBSA) in the medium was determined. Emulsified PBSA was consumed as a sole carbon source and an inducer of PCLE production by strain B47-9. Among the various concentrations of emulsified PBSA [0.09-0.9% (w/v)] used in flask cultivation, 0.27% yielded the maximum enzyme activity within a short cultivation period. To evaluate the residual concentration of emulsified PBSA in culture, emulsified PBSA in aliquots of culture supernatant was digested in vitro, and the concentration of released monomerised succinic acid was determined. Regardless of the initial concentration of emulsified PBSA in medium, PCLE activity was detected after residual succinic acid decreased below 0.04 mg/mL in culture broth. Jarfermentation was performed at a 0.27% PBSA concentration. Among the various airflow rates tested, 1 LPM resulted in a PCLE production rate of 1.0 U/mL/day. The enzyme activity in the resulting culture filtrate (4.2 U/2 mL) was shown to degrade commercial BP films (1 × 1 cm, 20 µm thickness) within 8 hours.

Control of seed dormancy and germination by DOG1-AHG1 PP2C phosphatase complex via binding to heme

Abscisic acid (ABA) regulates abiotic stress and developmental responses including regulation of seed dormancy to prevent seeds from germinating under unfavorable environmental conditions. ABA HYPERSENSITIVE GERMINATION1 (AHG1) encoding a type 2C protein phosphatase (PP2C) is a central negative regulator of ABA response in germination; however, the molecular function and regulation of AHG1 remain elusive. Here we report that AHG1 interacts with DELAY OF GERMINATION1 (DOG1), which is a pivotal positive regulator in seed dormancy. DOG1 acts upstream of AHG1 and impairs the PP2C activity of AHG1 in vitro. Furthermore, DOG1 has the ability to bind heme. Binding of DOG1 to AHG1 and heme are independent processes, but both are essential for DOG1 function in vivo. Our study demonstrates that AHG1 and DOG1 constitute an important regulatory system for seed dormancy and germination by integrating multiple environmental signals, in parallel with the PYL/RCAR ABA receptor-mediated regulatory system.The hormone abscisic acid (ABA) prevents seeds from germination when conditions are not suitable. Here the authors show that DOG1, a positive regulator of germination, impairs ABA signaling via genetic and physical interactions with the AHG1 phosphatase and that DOG1 binding to heme is required for this activity.