Stress-induced expression and structure of the putative gene hyp-1 for hypericin biosynthesis

The phenolic oxidative coupling protein (Hyp-1) with proposed activity in the biosynthesis of hypericin in Hypericum perforatum shares about 50 % sequence similarity with Bet.v.1-like/PR-10 proteins. In our previous study, we showed that this protein is not a limiting factor in hypericin biosynthesis. To ascertain the role of Hyp-1 in defense mechanisms, we have analyzed some structural features of the hyp-1 gene in 14 Hypericum species with different abilities to synthesise hypericin. We show that the hyp-1 gene possesses characteristics typical for genes encoding plant PR-10 proteins. The coding sequence of the hyp-1 gene is interrupted by a single 86- to 125-bp intron localised strictly in codon 62, which is a typical feature of the dicot PR-10 subfamily. The localisation of the intron is conserved in all 14 tested Hypericum species indicating a common evolutionary history with genes encoding PR-10 proteins. In addition, we report that the hyp-1 gene exhibits a similar response to stress conditions as the PR-10 proteins encoding genes. Following either wounding or infection by Agrobacterium tumefaciens, all analysed Hypericum species exhibited rapid and significant upregulation of hyp-1 gene expression; this was particularly observed in hypericin-producing species. On the other hand, in the presence of high levels of abscisic acid, different levels of gene expression were observed.     

The Activation of the Potato PR-10a Gene Requires the Phosphorylation of the Nuclear Factor PBF-1

The pathogenesis-related gene PR-10a (formerly STH[middot]2) is induced in various organs of potato after wounding, elicitor treatment, or infection by Phytophthora infestans. Deletion analysis of the promoter of the PR-10a gene enabled us to identify a 50-bp region, located between positions -155 and -105, necessary for the elicitor responsiveness of the [beta]-glucuronidase reporter gene in transgenic potato plants. Within this region, a 30-bp sequence, located between positions -135 and -105, was necessary for the activation of the promoter by the elicitor. However, strong promoter activity after elicitor treatment required the presence of a 20-bp sequence located between positions -155 and -135. The region between -135 and -105 was specifically recognized by two nuclear factors, PBF-1 (PR-10a Binding Factor 1) and PBF-2, and binding of PBF-1 was coordinated with the accumulation of the PR-10a mRNA. Gel shift assays using nuclear extracts pretreated with sodium deoxycholate or alkaline phosphatase suggested that PBF-1 is a multimeric factor in which at least one of the constituent proteins can be phosphorylated. Treatment with alkaline phosphatase also indicated that binding of PBF-1 is positively regulated by phosphorylation and that it is phosphorylated only in tissues in which PR-10a is expressed. The use of protein phosphatase and kinase inhibitors in vivo provided additional evidence that wounding and elicitor treatment induce the phosphorylation of PBF-1 and that this phosphorylation is associated with gene activation.     

Antimicrobial and Immunomodulatory Activities of PR-39 Derived Peptides

The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial- and immunomodulatory activity of PR-39, and N- and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-α production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics.     

Ethephon- and Jasmonate-Elicited Pathogenesis-Related Ribonucleases in Cultured Ginseng Cells

Cultured ginseng cells (Panax ginseng C.A. Mey strain R-1) produce two proteins exhibiting RNase activity (Pg1 and Pg2), which, on the basis of their amino acid sequences, have been earlier referred to intracellular pathogenesis-related proteins. An immunoenzyme technique for estimation of these proteins was developed. A close correlation was found between the content of these proteins and the RNase activity of the cultured cells. Ethephon and jasmonic acid activated the RNase activity, ethephon being more efficient. Salicylic acid did not activate Pg1 and Pg2; high concentrations of salicylic acid suppressed the RNase activity of the culture. The protein kinase inhibitor, H-7, reduced the content and activity of RNases both in the presence and absence of ethephon. The results obtained permit a suggestion that ethylene and jasmonic acid signaling pathways, which include protein phosphorylation, are involved in the induction of PR-10 proteins.     

Crystal structure of Hyp-1, a St. John’s wort protein implicated in the biosynthesis of hypericin

Hypericin, a red-colored naphtodianthrone, is a natural product synthesized in the medicinal plant Hypericum perforatum, widely known as St. John’s wort. Hypericin has been attracting a growing attention of the pharmaceutical industry because of its potential application in various therapies, including the treatment of depression. In vivo, hypericin is synthesized by dimerization of emodin in a complicated multistep reaction that is reportedly catalyzed by a small (17.8 kDa) protein, Hyp-1. Based on relatively low sequence similarity (～50%), Hyp-1 has been tentatively classified as a plant PR-10 (pathogenesis-related class 10) protein. Members of the PR-10 family are ubiquitous plant proteins associated with stress control and tissue differentiation but with no clearly understood molecular mechanism. They have, however, a well-defined folding canon, consisting of an extended antiparallel β-sheet wrapped around a C-terminal α-helix, enclosing in the protein interior a huge cavity, in which various hydrophobic ligands can be bound. Apart from Hyp-1, only two other PR-10 members have been found to possess enzymatic activity (S-norcoclaurine synthase and TcmN aromatase/cyclase). In this paper, we report a high-resolution crystal structure of Hyp-1, confirming that it indeed has a PR-10 fold. The protein binds multiple polyethylene glycol molecules, some of which occupy the hydrophobic cavity. The crystallographic model illustrates a high degree of conformational adaptability of both interacting partners for efficient binding. We have been unable, however, to dimerize emodin to hypericin using Hyp-1 as biocatalyst. This puzzling result does not have a clear explanation at this time.     

Lupinus luteus pathogenesis-related protein as a reservoir for cytokinin

Plant pathogenesis-related (PR) proteins of class 10 (PR-10) are small and cytosolic. The main feature of their three-dimensional structure is a large cavity between a seven-stranded antiparallel β-sheet and a long C-terminal α-helix. Although PR-10 proteins are abundant in plants, their physiological role remains unknown. Recent data have indicated ligand binding as their possible biological function. The article describes the structure of a complex between a classic PR-10 protein (yellow lupine LlPR-10.2B) and the plant hormone, trans-zeatin. Previously, trans-zeatin binding has been reported in a structurally related cytokinin-specific binding protein, which has a distant sequence relation with classic PR-10 proteins. In the present 1.35 Å resolution crystallographic model, three perfectly ordered zeatin molecules are found in the binding cavity of the protein. The fact that three zeatin molecules are bound by the protein when only a fourfold molar excess of the ligand was used indicates an unusual type of affinity for this ligand and suggests that LlPR-10.2B, and perhaps other PR-10 proteins as well, acts as a reservoir of cytokinin molecules in the aqueous environment of the cell.     

Spermine Is a Salicylate-Independent Endogenous Inducer for Both Tobacco Acidic Pathogenesis-Related Proteins and Resistance against Tobacco Mosaic Virus Infection

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.     

Selenium: potent stimulator of tyrosyl phosphorylation and activator of MAP kinase

Selenium, an essential biological trace element, is an integral component of several enzymes, and its use as a nutritional supplement has been popularized recently due to its potential role in low concentrations as an antioxidant and in higher concentrations as an anticancer agent. Selenium has also been reported to act as an insulin-mimetic agent with regard to normalization of blood glucose levels and regulation of some insulin-mediated metabolic processes. Little work, however, has been done concerning the pathway(s) by which this insulin-mimetic action occurs. In this study, we investigated the mechanism by which selenate exhibits insulin-mimetic properties in two different insulin responsive cell types, primary rat hepatocytes and 3T3 L1 adipocytes. We found that two proteins associated with the insulin signal cascade, the β-subunit of the insulin receptor and IRS-1, increased in tyrosyl phosphorylation in the presence of selenium. The third identified selenium activated signal protein, MAP kinase, has been implicated not only in the insulin signal transduction pathway but also in other growth factor-mediated responses. Using an in-gel activity assay for MAP kinase, we demonstrated that both the p42 and p44 MAP kinases are activated when either hepatocytes or adipocytes are incubated in the presence of selenate. In addition to the activation of these specific proteins, we found that selenium also eventually profoundly affected overall tyrosyl phosphorylation. Our results therefore show that selenium not only increased the phosphorylation of proteins identified in the insulin signal cascade but also affected the overall phosphorylation state of the cell.     

Expression of the ginseng PgPR10-1 in Arabidopsis confers resistance against fungal and bacterial infection

Korean ginseng (Panax ginseng C. A. Meyer) consists of nine cultivars from three Jakyung, Chungkyung, and Hwangsook lines. Among three previously identified PR-10 homologs from ginseng (PgPR10-1, PgPR10-2, and PgPR10-3), we found that the exact same sequence of PgPR10-2 exist in all tested nine cultivars. But a deletion and SNP was found in American ginseng (Panax quinquefolius). PR-10 proteins are known to be small and cytosolic, and showed similar three-dimensional structure. Here we show that the heterologous overexpression of PgPR10-1 in Arabidopsis showed enhanced resistance against Pseudomonas syringe, Fusarium oxysporum, and Botrytis cinerea and in-frame tagging with fluorescent protein showed its cytoplasm and nucleus localization. Protein–protein interaction of PgPR10-2 with PgPR10-1, PgPR10-2 and PgPR10-3 suggests that the PgPR10 proteins might form multimeric complexes in different cellular compartments to function in development and in defense-related mechanism. Differential response of PgPR10-1 and PgPR10-2 against different sets of biotic stresses in ginseng plant supports this notion.     

Kelch repeat proteins control yeast PKA activity in response to nutrient availability

Regulation of protein kinase A (PKA) by binding of cAMP to the regulatory subunit and the resulting release of the active catalytic subunit is a very well established mechanism of kinase activation. We have shown recently that PKA in budding yeast is also subject to an additional level of regulation that that modulates its activity in response to nutrient availability. Nutrient regulation of PKA activity requires a pair of proteins, Gpb1 and Gpb2, that contain several kelch repeats, a sequence motif that predicts that they fold into a β-propeller structure. The regulatory process mediated by Gpb1 and Gpb2 causes an increase in the stability and phosphorylation of the PKA regulatory subunit Bcy1 in response to low extracellular glucose concentrations. Phosphorylation of serine-145 of Bcy1 controls its stability, and other phosphorylation events at the cluster of serines at positions 74–84 correlate with changes in nutrient availability. Here we present data consistent with a model in which the effects of Gpb1 and Gpb2 on Bcy1 are an indirect consequence of their primary effects on the PKA catalytic subunits.Key words: protein kinase A, kelch repeat proteins, nutritional signaling, Saccharomyces cerevisiae     

Direct evidence for ribonucleolytic activity of a PR-10-like protein from white lupin roots

An abundant 17 kDa protein which was isolated and characterized from 10-day old healthy root tissue of white lupin (Lupinus albus) proved to have a high sequence similarity to pathogenesis-related proteins found in other species. Subsequently, a corresponding clone (LaPR-10) was identified in a cDNA library prepared from the same tissue that exhibited a high amino acid sequence similarity to a number of the PR-10 family proteins. The clone contains an open reading frame encoding a polypeptide of 158 amino acids, with a predicted molecular mass of 16905 Da and an isoelectric point of 4.66. Southern blot analysis indicates that LaPR-10 is likely a single-copy gene, or a member of a small gene family. The clone was expressed in Escherichia coli, and its protein product was purified to near homogeneity. Both the native and the recombinant proteins were immunorecognized by antibodies raised against pea PR-10 proteins, and exhibited a ribonucleolytic activity against several RNA preparations, including lupin root total RNA. Characterization of its enzymatic properties indicates that the LaPR-10 protein belongs to the class II ribonucleases. We present evidence that the white lupin 17 kDa protein is constitutively expressed during all stages of root development and, to a lesser extent, in other plant parts. In addition, we demonstrate the presence, in the LaPR-10 amino acid sequence, of a number of motifs that are common to most PR-10 proteins, as well as a RGD motif that is shared only with the alfalfa SRG1 sequence.     

Dimerization and protease resistance: New insight into the function of PR-1

The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15 kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants.     

The Comparative Analysis of Osmotins and Osmotin-Like PR-5 Proteins

Abstract: One of the ways that plants respond to biotic and/or abiotic stress factors is the accumulation of pathogenesis-related proteins of class 5 (PR-5), which are evolutionary conserved in the plant kingdom. Within the PR-5 family, a distinct subgroup of osmotin and closely related proteins has been characterized. In contrast to the extracellular forms of PR-5 proteins, osmotins presumably accumulate in the vacuole of the cell. They contain a C-terminal propeptide that is considered to be a determinant for vacuolar targeting. The comparison of the three-dimensional structure of tobacco PR-5 d with the sequences of some osmotins showed that the proteins consist of three conserved domains, with the acidic cleft between domains I and II. Besides the constitutive species and tissue-specific presence, the osmotins are also induced by several abiotic and biotic stresses. Among them, fungal infections can elicit osmotin gene expression, and most known proteins from the family have antifungal activity in in vitro assays. In agreement with the osmotin structure and data on the activity of similar proteins, a two-step mechanism, which involves reaction of osmotins with the fungal wall and the permeabilization of fungal membranes, is discussed.     

Phosphodiesterase activity and cyclic AMP content during early germination ofMucor rouxii spores

Incubation ofMucor rouxii spores in complex medium under aerobic conditions resulted in a very rapid transient increase in the level of soluble, low-K_m cAMP phosphodiesterase. Maximum activity was reached after 2–4 min. Simultaneously, the cAMP content increased quickly, reproducing the profile exhibited by phosphodiesterase. Activation of the enzymein vitro by a cAMP-dependent phosphorylation process showed that its stimulation was minimal at those times when its activity was highest. The correlation between cellular cAMP content, phosphodiesterase activity, and sensitivity of the enzyme to activation by phosphorylation suggests that thein vivo regulation of phosphodiesterase activity by cAMP-dependent phosphorylation may be the mechanism for shutoff of the cAMP signal elicited by glucose.