A region corresponding to second aspartate-rich motif in tryptophan isoprenylating enzyme, ComQ, serves as a substrate-binding site.

Posttranslational isoprenylation of a tryptophan residue identified from Bacillus quorum sensing pheromone, ComX pheromone, is unique and essential for the bioactivity. A modifying enzyme, ComQ, forms ComX pheromone from the ComX precursor and isoprenyl pyrophosphate and exhibits moderate similarity to isoprenyl pyrophosphate synthases. We investigated non-conserved region in ComQ, corresponding to isopentenyl pyrophosphate binding region of the synthases, using in vitro cell-free isoprenylation. These results suggested that the only conserved aspartic acid residue in the region of ComQ is critical for enzyme activity and responsible for ComX binding. Our findings should contribute to basic understanding of the mechanism of tryptophan isoprenylation.

Differential effects of the peptides Stomagen, EPF1 and EPF2 on activation of MAP kinase MPK6 and the SPCH protein level.

The positioning and density of leaf stomata are regulated by three secretory peptides, EPIDERMAL PATTERNING FACTOR 1 (EPF1), EPF2 and stomagen. Several lines of published evidence have suggested a regulatory pathway as follows. EPF1 and EPF2 are perceived by receptor complexes consisting of a receptor-like protein, TOO MANY MOUTHS (TMM), and receptor kinases, ERECTA (ER), ERECTA-LIKE (ERL) 1 and ERL2. These receptors activate a mitogen-activated protein (MAP) kinase module. MAP kinases phosphorylate and destabilize the transcription factor SPEECHLESS (SPCH), resulting in a decrease in the number of stomatal lineage cells. Stomagen acts antagonistically to EPF1 and EPF2. However, there is no direct evidence that EPF1 and EPF2 activate or that stomagen inactivates the MAP kinase cascade, through which they might regulate the SPCH level. Experimental modulation of these peptides in Arabidopsis thaliana would change the number of stomatal lineage cells in developing leaves, which in turn would change the expression of SPCH, making the interpretation difficult. Here we reconstructed this signaling pathway in differentiated leaf cells of Nicotiana benthamiana to examine signaling without the confounding effect of cell type change. We show that EPF1 and EPF2 are able to activate the MAP kinase MPK6, and that both EPF1 and EPF2 are able to decrease the SPCH level, whereas stomagen is able to increase it. Our data also suggest that EPF1 can be recognized by TMM together with any ER family receptor kinase, whereas EPF2 can be recognized by TMM together with ERL1 or ERL2, but not by TMM together with ER.

The second Phytophthora mating hormone defines interspecies biosynthetic crosstalk.

The heterothallic species of the agricultural pest Phytophthora use mating hormones α1 and α2 to regulate their sexual reproduction. Here we describe the absolute stereostructure of the second mating hormone α2 as defined by spectroscopic analysis and total synthesis. We have uncovered not only the interspecies universality of α hormones but also the pathway by which α2 is biosynthesized from phytol by A2-mating type strains and metabolized to α1 by A1 strains.

Anti-aging effects of hesperidin on Saccharomyces cerevisiae via inhibition of reactive oxygen species and UTH1 gene expression.

This study used a replicative lifespan assay of K6001 yeast to screen anti-aging food factors in commercial flavonoids. Hesperidin derived from the Citrus genus extended the lifespan of yeast at doses of 5 and 10 µM as compared with the control group (p<0.01, p<0.01). Reactive oxygen species (ROS), real-time PCR (RT-PCR), and lifespan assays of uth1 and skn7 mutants with the K6001 background were used to study the anti-aging mechanisms in yeast. The results indicate that hesperidin significantly inhibits the ROS of yeast, and UTH1 gene expression, and that SKN7 gene are involved in hesperidin-mediated lifespan extension. Further, increases in the Sir2 homolog, SIRT1 activity, and SOD gene expression were confirmed at doses of 5 (p<0.01) and 10 µM (p<0.05). This suggests that Sir2, UTH1 genes, and ROS inhibition after administration of hesperidin have important roles in the anti-aging effects of yeast. However, the aglycon hesperetin did not exhibit anti-aging effects in yeast.

Termitomycesphins G and H, additional cerebrosides from the edible Chinese mushroom Termitomyces albuminosus.

Two new cerebrosides, termitomycesphins G and H, were isolated from the edible Chinese mushroom, Termitomyces albuminosus (Berk.) Herm., and exhibited neuritogenic activity against PC12 cells. Their structures and absolute stereochemistry were elucidated by spectroscopic methods and by a comparison of the specific rotation of the hydrogenated products from termitomycesphins H and C. These cerebrosides possessed a unique modification by a hydroxyl group at the middle of the long-chain base, like earlier congeners termitomycesphins A-F. Termitomycesphin G with a 16-carbon-chain fatty acid showed higher neuritogenic activity than that of termitomycesphin H with an 18-carbon-chain fatty acid. This effect was observed within the termitomycesphins, suggesting that the chain length of the fatty acyl moiety played a key role in the neuritogenic activity.

Lack of the consensus sequence necessary for tryptophan prenylation in the ComX pheromone precursor.

ComX, an oligopeptide pheromone that stimulates the natural genetic competence controlled by quorum sensing in Bacillus subtilis and related bacilli, contains a prenyl-modified tryptophan residue. Since ComX is the only protein known to contain prenylated tryptophan, the universality of this unique posttranslational modification has yet to be determined. Recently, we developed a cell-free assay system in which the tryptophan residue in the ComX(RO-E-2) pheromone precursor derived from B. subtilis strain RO-E-2 can be geranylated by the ComQ(RO-E-2) enzyme. We report here our attempt to identify the consensus sequence surrounding the geranylated tryptophan residue by using the cell-free system with various ComX(RO-E-2) pheromone precursor analogs. We found that [47-58]ComX(RO-E-2), corresponding to the C-terminal 12-residue peptide of the pheromone precursor, contained a short sequence essential for geranylation. We also found that the length of the sequence between the tryptophan residue and the C-terminus was important for geranylation, and that some [47-58]ComX(RO-E-2) pheromone precursor amino acids were involved in the geranylation reaction. However, we could not identify a consensus sequence surrounding the geranylated tryptophan. Our evidence suggests that, like Rab which lacks a consensus sequence yet is geranylgeranyl-modified on a cysteine residue, the ComX pheromone and its precursor also lack a consensus sequence.

Analogs of the CLV3 peptide: synthesis and structure-activity relationships focused on proline residues.

CLAVATA3 (CLV3) is a plant peptide hormone in which the proline residues are post-translationally hydroxylated and glycosylated. CLV3 plays a key role in controlling the stem cell mass in the shoot meristem of Arabidopsis thaliana. In a previous report, we identified a dodecapeptide (MCLV3) from CLV3-overexpressing Arabidopsis calli; MCLV3 was the smallest functional peptide derived from the CLV3 precursor. Here, we designed a series of MCLV3 analogs in which proline residues were substituted with proline derivatives or N-substituted glycines (peptoids). Peptoid substitution at Pro9 decreased bioactivity without affecting specific binding to the CLV1-related protein in cauliflower membrane. These findings suggest that peptoid-substituted peptides would be lead compounds for developing potential agonists and antagonists of CLV3.

The geranyl-modified tryptophan residue is crucial for ComXRO-E-2 pheromone biological activity.

The ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in gram-positive bacteria, Bacillus. We have reported the structure of the ComX(RO-E-2) pheromone, which is produced by the RO-E-2 strain of Bacillus subtilis. ComX(RO-E-2) analogs with substituted amino acids and isoprenoid modified tryptophan residues (e.g., prenyl, geranyl, and farnesyl), were synthesized and examined for biological activity. These results indicate that Phe-Trp(∗)(Ger)-NH(2) is the minimum pharmacophore of the ComX(RO-E-2) pheromone. Furthermore, the length of the isoprenoid moiety (i.e., modification style), and the presence of double bonds, are crucial for biological activity. The modification style of the ComX pheromone is more important than the peptide sequence with respect to biological activity.

Anti-aging effects of phloridzin, an apple polyphenol, on yeast via the SOD and Sir2 genes.

The anti-aging effects of phloridzin on the yeast Saccharomyces cerevisiae were investigated by employing a replicative lifespan assay of the K6001 yeast strain. After administrating phloridzin at doses of 3, 10, and 30 µM, the lifespan of the yeast was significantly prolonged in comparison with the untreated group (p<0.01, p<0.001). To determine the mechanism of action, anti-oxidative experiments and ROS assay were performed. Phloridzin improved the viability of the yeast dose-dependently under oxidative stress by 7.5 mM H(2)O(2), and a low dose of phloridzin inhibited ROS of the yeast. Further, SOD1, SOD2, and Sir2 gene expression was examined by reverse transcription-polymerase chain reaction (RT-PCR), and was found to be significantly increased. Finally, superoxide dismutase (SOD) and SIRT1 activity assays showed that phloridzin notably increased the activity of SOD and SIRT1. These results suggest that SOD and Sir2 have important roles in phloridzin-regulated lifespan extension of yeast, and potentially anti-aging effects for mammalian cells via SIRT1.

New neuritogenic steroidal saponin from Ophiopogon japonicus (Thunb.) Ker-Gawl.

A new steroidal saponin was isolated from Ophiopogon japonicus. This saponin possesses a modification by 2-hydroxy-3-methylvalerylation of the hydroxyl group at C-4' of the sugar, linked to C-1 of the aglycone. It exhibited significant neuritogenic activity for PC12 cells. The structure-activity relationship revealed the aglycone, rather than the sugar moieties and acylation, to be important for the neuritogenic activity.

Peptide hormones in plants.

In recent years, numerous biochemical and genetic studies have demonstrated that peptide signaling plays a greater than anticipated role in various aspects of plant growth and development. A substantial proportion of these peptides are secretory and act as local signals mediating cell-to-cell communication. Specific receptors for several peptides were identified as being membrane-localized receptor kinases, the largest family of receptor-like molecules in plants. These findings illustrate the importance of peptide signaling in the regulation of plant growth, functions that were previously ascribed to the combined action of small lipophilic compounds referred to as "traditional plant hormones." Here, we outline recent advances in the current understanding of biologically active peptides in plants, currently regarded as a new class of plant hormones.

Stomatal density is controlled by a mesophyll-derived signaling molecule.

Stomata are composed of a pair of guard cells and a pore between them, and their density and positions are regulated by developmental and environmental signals. In a screen in which we overexpressed many genes coding for putative secretory proteins one by one in Arabidopsis, we identified a gene named STOMAGEN, which increases stomatal density when overexpressed. The STOMAGEN gene encodes a small peptide with a putative secretory signal sequence at its N-terminus and is expressed preferentially in mesophyll cells. This peptide belongs to the EPIDERMAL PATTERNING FACTOR (EPF) family of the cysteine-rich peptides superfamily. The mature form was a 45-amino-acid peptide (stomagen) with three intramolecular disulfide bonds. Stomagen treatment at very low concentrations, as low as 10 nM, increased the stomatal density of wild-type Arabidopsis plants. We propose that stomagen is a mesophyll-to-epidermis signaling molecule that positively regulates stomatal density. We also suggest that stomagen increases stomatal density by competing with negative regulators EPF1 and EPF2 for the receptor-like protein TOO MANY MOUTHS.

Synthesis and absolute configuration of hormone alpha1.

An important biological event in phytopathogens of the genus Phytophthora is sexual reproduction, which is conducted by two mating types, A1 and A2. A factor known as hormone alpha1 is secreted by the A1 mating type and induces the formation of sexual spores (oospores) in the A2 mating type. Here we describe the asymmetric synthesis and assignment of the absolute configuration of hormone alpha1 by oospore-inducing assays of the synthesized isomers.

Dual assay for MCLV3 activity reveals structure-activity relationship of CLE peptides.

The dodecapeptide MCLV3 is a functional peptide, derived from the CLV3 precursor protein, which is a candidate ligand of the CLV1/CLV2 receptor complex that restricts the stem cell population in the shoot apical meristem (SAM). MCLV3 can induce shoot and root meristem consumption, the typical phenotype of transgenic plants overexpressing CLV3. We investigated the bioactivities of a series of alanine-substituted MCLV3 and related peptides on the root growth of Arabidopsis. The structure-activity relationship (SAR) of MCLV3 had high similarity with that of tracheary element differentiation inhibitory factor (TDIF). We also evaluated the binding activities of the peptides by a competitive receptor binding assay using tritiated MCLV3 and the membrane fraction of a tobacco BY-2 cell line overexpressing the MCLV3 ectodomain. This dual assay, combining a biological and receptor binding assay for evaluating the activities of MCLV3-related peptides, uncovered the SAR of MCLV3, and indicated that the terminal residues play critical roles in exerting its activity and are important for specific binding to the receptor, CLV1.

Chemical structure of posttranslational modification with a farnesyl group on tryptophan.

Bacillus subtilis and related bacilli produce a posttranslationally modified oligopeptide, the ComX pheromone, that stimulates natural genetic competence controlled by quorum sensing. The ComX(RO-C-2) pheromone from strain RO-C-2 must be modified with a farnesyl group on the Trp residue, but the precise structure is not known. Here we report the precise nature of posttranslational farnesylation of ComX(RO-C-2) pheromone on the Trp residue, resulting in the formation of a tricyclic structure. The ComX(168) pheromone, produced by the standard laboratory strain used in the study of B. subtilis, is also posttranslationally farnesylated according to phylogenetic resemblance.

Articulospora sp. produces Art1, an inhibitor of bacterial histidine kinase.

A two-component system (TCS) comprising a histidine kinase (HK) sensor and a response regulator (RR) plays important roles in regulating the virulence of many pathogenic bacteria. We used a new screening method to isolate novel inhibitor Art1 against bacterial sensory HK from an acetone extract of solid cultures of Articulospora sp., an aquatic hypomycete. Art1 inhibited the ATP-dependent autophosphorylation of recombinant glutathione S-transferase-fusion protein SasA, a cyanobacterial HK, with an IC50 value of 9.5 microg/ml.

Peptide signalling in plants.

Peptide signals play crucial roles in all aspects of the plant life cycle. An understanding of peptide signal production and reception mechanisms is beginning to emerge. Studies on the signal-transduction cascades that follow the reception of peptide signals are just beginning.

Biosynthetic studies on a myxobacterial antibiotic, cystothiazole A: biosynthetic precursors of the carbon skeleton.

Biosynthetic studies on cystothiazole A (1), a beta-methoxyacrylate-type antifungal compound from a myxobacterium, were performed by feeding the producing organism, Cystobacter fuscus, with stable-isotope-labeled compounds including [2-(13)C]acetate, [1,2-(13)C2]acetate, [1-(13)C]propionate, L-[1-(13)C]serine, L-[methyl-(13)C]methionine, and DL-valine-d8. The polyketide moiety of 1 was found to be derived from acetate and propionate, the bithiazole moiety from L-serine, the O-methyl groups from the S-methyl group of L-methionine, and the isopropyl moiety from L-valine, which should be the metabolic precursor of isobutyryl-CoA.

New haliangicin isomers, potent antifungal metabolites produced by a marine myxobacterium.

Haliangicin is a beta-methoxyacrylate-type polyene antibiotic isolated from the unique marine myxobacterium Haliangium ochraceum. A further investigation of the extract of this microorganism has resulted in the isolation of haliangicin as well as its geometrical isomers. The configuration of the epoxide in haliangicin, which was unknown previously, was determined in this study. The planar structures with partial stereochemistry of these isomers were elucidated by spectroscopic analyses. The major isomer, cis-haliangicin, is a cis isomer of the epoxide portion of haliangicin. Haliangicins B approximately D are geometrical isomers of the polyene moiety, and present as an inseparable mixture of cis and trans epoxide isomers. Their antifungal activities were also evaluated in comparison with haliangicin.