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.

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.

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.

Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system.

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.

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.

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.

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.

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.

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.

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.

Involvement of phytosulfokine in the attenuation of stress response during the transdifferentiation of zinnia mesophyll cells into tracheary elements.

Phytosulfokine (PSK) is a sulfated peptide hormone required for the proliferation and differentiation of plant cells. Here, we characterize the physiological roles of PSK in transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans 'Canary Bird') into tracheary elements (TEs). Transcripts for a zinnia PSK precursor gene, ZePSK1, show two peaks of expression during TE differentiation; the first accumulation is transiently induced in response to wounding at 24 h of culture, and the second accumulation is induced in the final stage of TE differentiation and is dependent on endogenous brassinosteroids. Chlorate, a potent inhibitor of peptide sulfation, is successfully applied as an inhibitor of PSK action. Chlorate significantly suppresses TE differentiation. The chlorate-induced suppression of TE differentiation is overcome by exogenously applied PSK. In the presence of chlorate, expression of stress-related genes for proteinase inhibitors and a pathogenesis-related protein is enhanced and changed from a transient to a continuous pattern. On the contrary, administration of PSK significantly reduces the accumulation of transcripts for the stress-related genes. Even in the absence of auxin and cytokinin, addition of PSK suppresses stress-related gene expression. Microarray analysis reveals 66 genes down-regulated and 42 genes up-regulated in the presence of PSK. The large majority of down-regulated genes show significant similarity to various families of stress-related proteins, including chitinases, phenylpropanoid biosynthesis enzymes, 1-aminocyclopropane-1-carboxylic acid synthase, and receptor-like protein kinases. These results suggest the involvement of PSK in the attenuation of stress response and healing of wound-activated cells during the early stage of TE differentiation.

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.

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.

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.

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.

Arabidopsis CLV3 peptide directly binds CLV1 ectodomain.

CLV1, which encodes a leucine-rich repeat receptor kinase, and CLV3, which encodes a secreted peptide, function in the same genetic pathway to maintain stem cell populations in Arabidopsis shoot apical meristem. Here, we show biochemical evidence, by ligand binding assay and photoaffinity labeling, that the CLV3 peptide directly binds the CLV1 ectodomain with a dissociation constant of 17.5 nM. The CLV1 ectodomain also interacts with the structurally related CLE peptides, with distinct affinities depending on the specific amino acid sequence. Our results provide direct evidence that CLV3 and CLV1 function as a ligand-receptor pair involved in stem cell maintenance.

Spongolactams, farnesyl transferase inhibitors from a marine sponge: isolation through an LC/MS-guided assay, structures, and semisyntheses.

Novel nitrogenous diterpenoids, spongolactams A-C (1-3), were isolated as trace components of an Okinawan marine sponge, Spongia sp., by an LC/MS-guided assay for farnesyl transferase (FTase) inhibitors. Their structures were elucidated by spectroscopic analyses. To evaluate their structures and biological activity, the metabolites were semisynthesized from the known furanoditerpene 5, obtained from the same sponge. Three related compounds 4, 13, and 16 were also semisynthesized. The IC50 values against FTase for 1-3 were 23, 130, and >260 microM, respectively, while the IC50 values against a human tumor cell line were 2.0, 3.5, and 20 microM, respectively. The structure-activity relationships within the six compounds suggest some positive correlation between FTase inhibitory and cytotoxic activities.