Study of a new group of bioregulators isolated from the greater plantain (<Emphasis Type="Italic">Plantago major</Emphasis> L.)

Proteins with physicochemical properties and biological activity similar to those of membranotropic homeostatic tissue-specific bioregulators that had been found earlier in various animal tissues were discovered in leaves of the common plantain (Plantago major L.). To study the specific activity of these plant proteins, we developed an experimental model for organotypic roller cultivation of newt (Pleurodeles waltl) skin tissue in vitro. We showed that the plant proteins of interest exert the wound-healing effect, which is characteristic of this plant, on the skin of vertebrates both in vitro and in vivo.     

Temporal generation of multiple antifungal proteins in primed seeds

A drastic increase of antifungal activity was demonstrated during plant seed germination and in seed protein extract in vitro. Multiple antifungal proteins with a wide spectrum of activity were generated and identified. Chromatographic and electrophoretic analysis demonstrated that during seed germination, more fractions with potent antifungal activity were generated, and the antifungal activity shifted from small molecules to high molecular proteins. This germination-related increase of antifungal activity were observed in all three plants tested, i.e., cheeseweed, cigar tree and wheat. This rapid increase of antifungal activity was also observed with incubation of seed proteins in vitro, suggesting that at least part of the antifungal protein generation is independent of gene expression. Seven antifungal proteins with activities against five different plant pathogens were isolated from the active fractions. However, random digestion of purified seed protein with multiple proteinases failed to generate any antifungal proteins. It is suggested that during plant seed germination, a regulated biochemical process takes place that results in the generation of multiple peptides or proteins with antifungal activities. This onset of antifungal proteins is transitional in nature, but could play an important role in the protection of plants in early stage of development when the more sophisticated defense system has yet to develop.     

Skin cell induction of calcitonin gene-related peptide in embryonic sensory neurons in vitro involves activin

Target skin cells induce the neuropeptide calcitonin gene-related peptide (CGRP) in na?ve embryonic dorsal root ganglion (DRG) neurons in vitro, but the molecular basis of that induction is not known. Recombinant activin or bone morphogenetic proteins (BMPs) dramatically increase the number of sensory neurons with CGRP and substance P in vitro (X. Ai et al., 1999, Mol. Cell. Neurosci. 14, 506-518). These experiments were designed to test if activin or BMPs accounted for the CGRP-inductive activity by skin cells. To identify factors from skin that induce CGRP, we developed a bioassay in which embryonic DRG neurons isolated before peripheral target contact in vivo are challenged in vitro with specific factors. Conditioned medium from an embryonic rat skin cell line induced neuronal CGRP expression, and induction was blocked by follistatin, implicating transforming growth factor family members. Immunoblot analysis revealed that the skin cell line medium contained several activin and bone morphogenetic protein moieties. Antibody specific to activin neutralized most of the CGRP-inductive activity in skin conditioned medium. These data indicate that the CGRP-inductive action of skin cells involves activin and establish activin as a candidate regulator of this sensory neuropeptide phenotype during development.     

Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif

Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates. Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the [alpha] subunit of yeast Rab GGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity.     

Differential antifungal and calcium channel-blocking activity among structurally related plant defensins

Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invading fungi. Structures of several plant defensins share a Cys-stabilized alpha/beta-motif. Structural determinants in plant defensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca(2+). By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity reside in the carboxy-terminal region (amino acids 31-45) of MsDef1. We further define the active site by demonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for the first time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca(2+) channel in a manner akin to a virally encoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca(2+) channel. From these results, we speculate that the two unrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas the two structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.     

Effect of water stress on the photosynthetic apparatus of plants and the protective role of cytokinins: a review

Characteristics of photosynthetic apparatus (the pool of pigments and proteins; the activity of photosystems; the intensities of in vivo photoassimilation of carbon dioxide and in vitro activity of enzymes of carbon metabolism; leaf structure; chloroplast structure), undergoing changes under the conditions of water deficiency, have been reviewed. The protective role of cytokinins is due to their regulatory effects on the renewal of disrupted cellular structures, the condition of the stomata, and de novo synthesis and activation of proteins that are required for increasing plant resistance to water stress.     

Analysis of the in planta antiviral activity of elderberry ribosome-inactivating proteins.

Although the type-2 ribosome-inactivating proteins (SNA-I, SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are all devoid of rRNA N-glycosylase activity towards plant ribosomes, some of them clearly show polynucleotide-adenosine glycosylase activity towards tobacco mosaic virus RNA. This particular substrate specificity was exploited to further unravel the mechanism underlying the in planta antiviral activity of ribosome-inactivating proteins. Transgenic tobacco (Nicotiana tabacum L. cv Samsun NN) plants expressing the elderberry ribosome-inactivating proteins were generated and challenged with tobacco mosaic virus in order to analyze their antiviral properties. Although some transgenic plants clearly showed antiviral activity, no clear correlation was observed between in planta antiviral activity of transgenic tobacco lines expressing the different ribosome-inactivating proteins and the in vitro polynucleotide-adenosine glycosylase activity of the respective proteins towards tobacco mosaic virus genomic RNA. However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mechanism on the virus. In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins.     

Peptide Fragments From Plant Vicilins Expressed in <Emphasis Type="Italic">Escherichia Coli</Emphasis> Exhibit Antimicrobial Activity <Emphasis Type="Italic">In Vitro</Emphasis>

Peptide fragments that exhibit antimicrobial activity in vitro have been shown to be produced by cleavage from the hydrophilic region near the N terminus of various vicilin proteins in plant seeds. Three peptide sequences identified in the hydrophilic region of vicilin seed proteins of Macadamia integrifolia and Theobroma cacao were predicted to exhibit antimicrobial activity based on sequence similarity to antimicrobial peptides that had been previously purified from macadamia kernels. Histidine-tagged versions of the putative antimicrobial peptides were expressed in Escherichia coli, purified, and demonstrated to have in vitro antimicrobial activity. There are many vicilin sequences in the growing plant genome sequence databases, and this expression method provides a high-throughput process for functionally testing the potential of internal peptide fragments of vicilins as novel antimicrobial molecules.     

Grass group I pollen allergens (beta-expansins) lack proteinase activity and do not cause wall loosening via proteolysis.

Group I grass pollen allergens make up a subgroup of the beta-expansin family of cell wall loosening proteins in plants. A recent study reported that recombinant Phl p 1, the group I allergen from timothy grass pollen, was associated with papain-like proteinase activity and suggested that expansins loosen the plant cell wall via proteolysis. We tested this idea with three experimental approaches. First, we evaluated three purified native group I allergens from timothy grass, ryegrass and maize (Phl p 1, Lol p 1, Zea m 1) using five proteinase assays with a variety of substrates. The proteins had substantial wall loosening activity, but no detectable proteolytic activity. Thus we cannot confirm proteolytic activity in the pollen allergen class of beta-expansins. Second, we tested the ability of proteinases to induce cell wall extension in vitro. Tests included cysteine proteinases, serine proteinases, aspartic proteinases, metallo proteinases, and aggressive proteinase mixtures, none of which induced wall extension in vitro. Thus, wall proteins are unlikely to be important load-bearing components of the plant cell wall. Third, we tested the sensitivity of beta-expansin activity and native wall extension activity to proteinase inhibitors. The results show that a wide range of proteinase inhibitors (phenylmethanesulfonyl fluoride, N-ethylmaleimide, iodoacetic acid, Pefabloc SC, and others) inhibited neither activity. From these three sets of results we conclude proteolysis is not a likely mechanism of plant cell wall loosening and that the pollen allergen class of beta-expansins do not loosen cell walls via a proteolytic mechanism.     

Importance of tryptophan nitration of carbonic anhydrase III for the morbidity of atopic dermatitis

The nitration of proteins results from the vigorous production of reactive nitrogen species in inflammatory disease. We previously reported the proteomic analysis of nitrated tryptophan residues in in vitro model cells for inflammatory diseases using a 6-nitrotryptophan-specific antibody. In this paper, we applied this method to the analysis of a disease model animal and identified the 6-nitrotryptophan-containing proteins in the skin of atopic dermatitis model mice (AD-NC/Nga mice). We found three nitrotryptophan-containing proteins, namely, carbonic anhydrase III (CAIII), α-enolase (α-ENO), and cytoskeletal keratin type II (KTII), and identified the positions of the nitrotryptophan residues in their amino acid sequences: Trp47 and Trp123 in CAIII, Trp365 in α-ENO, and Trp221 in KTII. Among these, the nitration of CAIII was increased not only in the lesional skin of AD-NC/Nga mice but also in the mice that did not present any symptoms. The in vitro nitration of purified CAIII by peroxynitrite reduced its CO₂ hydratase activity in a dose-dependent manner. In addition, we found that CAIII was induced during the differentiation of normal human epidermal keratinocytes. Furthermore, we found the presence of CAIII and the formation of 6-nitrotryptophan-containing proteins in both the lesional and the nonlesional sections of the skin of patients with atopic dermatitis through immunohistochemical staining. This study provides the first demonstration of the formation of 6-nitrotryptophan in human tissues and disease.     

The E6 and E7 proteins of the cutaneous human papillomavirus type 38 display transforming properties

Several studies have suggested the involvement of cutaneous human papillomaviruses (HPVs) in the development of nonmelanoma skin cancers. Here we have characterized the in vitro properties of E7 proteins of three cutaneous HPV types, 10, 20, and 38, which are frequently detected in skin specimens. We show that HPV38 E7 is able to inactivate the tumor suppressor pRb and induces loss of G(1)/S transition control, a key event in carcinogenesis. In contrast, HPV10 and HPV20 E7 proteins do not display these in vitro transforming activities. We also show that the two early proteins E6 and E7 of HPV38 are sufficient to corrupt the cell cycle and senescence programs in primary cells, inducing active and long-lasting proliferation of primary human keratinocytes, the natural host cells. Our study shows that E6 and E7 of this cutaneous HPV type have transforming activity in primary human cells, suggesting a role for HPV38 infection in skin carcinogenesis. In further support of such a role, we detected HPV38 DNA in approximately 50% of nonmelanoma skin cancers, but only in 10% of healthy skin specimens (P < 0.001).     

Berberine inhibits TPA-induced MMP-9 and IL-6 expression in normal human keratinocytes

Berberine is a plant ingredient that has anti-inflammatory and anti-oxidative effects. Matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) are known to be highly induced by ultraviolet (UV) light and may play important roles in UV-induced skin inflammation and the skin aging process. In this study, we investigated the effects of berberine on MMP-9 and IL-6 expression in normal human keratinocytes (NHK). Our results demonstrated that berberine dose-dependently inhibited basal and TPA-induced expression and activity of MMP-9, and also suppressed TPA-induced IL-6 expression. Berberine prevented TPA-induced ERK activation and AP-1 DNA binding activity. Therefore, berberine may be used as an effective ingredient for anti-skin aging products, which can prevent skin inflammation and the degradation of extracellular matrix proteins, including collagen, by MMPs.     

A new class of proteins capable of binding transition metals

Ion uptake, transport, and sequestration are essential to meet the nutritional requirements for plant growth and development. Furthermore, regulation of these processes is critical for plants to tolerate toxic levels of ions. The examination of isoprenylated proteins encoded by Arabidopsis thaliana and Glycine max cDNAs revealed a unique family of proteins containing putative metal-binding motifs (the core sequence is M/LXCXXC). Here, we describe this new class of proteins, which are capable of being isoprenylated and binding transition metal ions. Members of this family contain consensus isoprenylation (CaaX) sites, which we demonstrate are efficiently isoprenylated in vitro. ATFP3, a representative of the Arabidopsis family, was expressed in Escherichia coli and examined for metal-binding activity in vitro. Analysis of the interaction of ATFP3 with metal-chelating columns (IMAC) suggested that it binds to Cu²⁺, Ni²⁺, or Zn²⁺. To test whether proteins with these characteristics are present in other plant species, tobacco BY2 cells were labeled in vivo with [¹⁴C]mevalonate and the resulting mevalonate-labeled proteins were tested for metal-binding activity. Several soluble, isoprenylated proteins which bound copper-IMAC columns were revealed. Consistent with a wide-spread distribution of these proteins in plants, their presence was observed in Arabidopsis, soybean, and tobacco.     

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate on newly synthesized proteins in mouse epidermis

We have analyzed the effects of treatment of mouse epidermis with the potent tumor promoter TPA on the profile of newly synthesized proteins. TPA was applied to the skin of the intact mouse, and either 3 or 24 hr later skin fragments were pulse-labeled in vitro with ³⁵S-methionine for 4 hr. The epidermal proteins were extracted and separated by two-dimensional gel electrophoresis. Over 200 individual proteins were resolved in acidic gels. At least 10 of these showed major (by a factor of 5 or more) increases or decreases in response to TPA; eight of these appear to be keratin proteins. Two-dimensional gel profiles of basic proteins synthesized by mouse epidermis resolved over 100 individual proteins. Only one of these showed a significant change in response to TPA. This 41 kd protein increased more than 100-fold within 24 hr after the application of TPA. Treatment of mouse skin with mezerein, a plant diterpene structurally related to TPA, produces an almost identical change in the pattern of proteins produced. Four agents that induce hyperplasia but are not potent tumor promoters, ethylphenylpropiolate, acetic acid, turpentine oil and the Ca⁺⁺ ionophore A23187, modulate the synthesis of only three of the keratin proteins. Thus the changes in protein profiles induced by TPA and mezerein are not simply the consequence of hyperplasia. In addition, application to mouse skin of a glucocorticoid that is a potent inhibitor of tumor promotion inhibits most of the changes in protein profiles induced by TPA. Taken together, these results indicate that TPA and mezerein induce early and marked changes in the profile of specific epidermal proteins. It seems likely that some of these changes are directly related to the process of tumor promotion.     

Existence of a plant tyrosylprotein sulfotransferase: novel plant enzyme catalyzing tyrosine O-sulfation of preprophytosulfokine variants in vitro

An in vitro assay system to detect tyrosylprotein sulfotransferase (TPST) activity of higher plant cells was established, using synthetic oligopeptides based on the deduced amino acid sequence of a phytosulfokine-alpha (PSK-alpha) precursor. TPST activity was found in microsomal membrane fractions of rice, asparagus and carrot cells and it was confirmed that acidic amino acid residues adjacent to the tyrosine residues of acceptor peptides were essential to the sulfation reaction. The asparagus TPST exhibited a broad pH optimum of 7.0-8.5, required manganese ions for maximal activity and appeared to be a membrane-bound protein localized in the Golgi apparatus. These enzymes should be defined as a new class of plant sulfotransferases that catalyze tyrosine O-sulfation of a PSK-alpha precursor and other unknown proteins.     

Overexpression of Arabidopsis AGD7 causes relocation of Golgi-localized proteins to the endoplasmic reticulum and inhibits protein trafficking in plant cells

ADP ribosylation factor (Arf) GTPase-activating proteins (GAPs) promote the hydrolysis of GTP bound to Arfs to GDP, which plays a pivotal role in regulating Arfs by converting the active GTP-bound forms of these proteins into their inactive GDP-bound forms. Here, we investigated the biological role of AGD7, an Arf GAP homolog, in Arabidopsis (Arabidopsis thaliana). We show that AGD7 bears a highly conserved N-terminal region and a unique C-terminal region, interacts with Arf1 both in vitro and in vivo, and stimulates Arf1 GTPase activity in a phosphatidic acid-dependent manner in vitro. In plant cells, AGD7 localized to the Golgi complex, where its overexpression was found to inhibit the Golgi localization of gamma-subunit of coat proteins and promote the relocation of Golgi proteins into the endoplasmic reticulum in both protoplasts and transgenic plants. Furthermore, overexpression of AGD7 inhibited anterograde trafficking of proteins from the endoplasmic reticulum. We propose that AGD7 functions as a GAP for Arf1 in the Golgi complex and plays a critical role in protein trafficking by controlling Arf1 activity.     

Differentiation of varicella-zoster virus ORF47 protein kinase and IE62 protein binding domains and their contributions to replication in human skin xenografts in the SCID-hu mouse

To investigate the role of the ORF47 protein kinase of varicella-zoster virus (VZV), we constructed VZV recombinants with targeted mutations in conserved motifs of ORF47 and a truncated ORF47 and characterized these mutants for replication, phosphorylation, and protein-protein interactions in vitro and for infectivity in human skin xenografts in the SCID-hu mouse model in vivo. Previous experiments showed that ROka47S, a null mutant that makes no ORF47 protein, did not replicate in skin in vivo (J. F. Moffat, L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin, Proc. Natl. Acad. Sci. USA 95:11969-11974, 1998). The construction of VZV recombinants with targeted ORF47 mutations made it possible to assess the effects on VZV infection of human skin xenografts of selectively abolishing ORF47 protein kinase activity. ORF47 mutations that resulted in a C-terminal truncation or disrupted the DYS kinase motif eliminated ORF47 kinase activity and were associated with extensive nuclear retention of ORF47 and IE62 proteins in vitro. Disrupting ORF47 kinase function also resulted in a marked decrease in VZV replication and cutaneous lesion formation in skin xenografts in vivo. However, infectivity in vivo was not blocked completely as long as the capacity of ORF47 protein to bind IE62 protein was preserved, a function that we identified and mapped to the N-terminal domain of ORF47 protein. These experiments indicate that ORF47 kinase activity is of critical importance for VZV infection and cell-cell spread in human skin in vivo but suggest that it is the formation of complexes between ORF47 and IE62 proteins, both VZV tegument components, that constitutes the essential contribution of ORF47 protein to VZV replication in vivo.