Rossella Miele

The synthesis of antimicrobial peptides in the skin of Rana esculenta is stimulated by microorganisms

Secretions of amphibian skin glands contain numerous antimicrobial peptides that play a crucial role in the defense against microorganisms. The location of these glands on the surface of the animal makes them a useful model for in vivo studies of the relationships between the innate immune system and the natural flora. Here, we present the results of a study showing that in Rana esculenta the total antimicrobial activity of skin secretion is modulated by the presence of the natural flora. Frogs kept in a sterile environment do not produce antimicrobial peptides. This finding represents the first in vivo demonstration of the induction of defense peptides in a vertebrate. We also present data on the morphological changes in skin glands of animals kept in sterile conditions or treated with glucocorticoids. Cells from glands regenerated under normal conditions, but not those from “sterile” frogs, contain κB‐binding activity in the cytoplasm.

Molecular cloning of a bombinin gene from Bombina orientalis: detection of NF-κB and NF-IL6 binding sites in its promoter

The sequence of a gene from Bombina orientalis was determined which codes for antibacterial peptides. The gene comprises two exons separated by a large intron. Exon 1 codes for the signal peptide, while exon 2 contains the genetic information for two identical bombinins and one bombinin H. The promoter region of the bombinin gene contains putative recognition sites for nuclear factors, such as NFκB and NF‐IL6. In vivo experiments on B. orientalis have shown that a short contact with bacteria is sufficient to induce a marked increase in the amount of antibacterial peptides in the skin secretion of frogs. This increase was suppressed by pretreatment with glucocorticoids. In the latter case, a significant increase of IκBα in the secretion is also detectable.

Effect of glucocorticoids on the synthesis of antimicrobial peptides in amphibian skin

Gene‐encoded peptide antibiotics are widespread in insects, plants and vertebrates and confer protection against bacterial and fungal infections. NF‐κB is an important transcription factor for many immunity‐related mammalian proteins and also for insect immune genes. The activity of NF‐κB is regulated by the interaction with an inhibitor, IκB. It was recently demonstrated that glucocorticoids induce the synthesis of IκB in human cell lines. So far, all genes for peptide antibiotics have promoter motifs with NF‐κB binding sites, but its actual function in peptide regulation has been studied only in insects. Here we show that glucocorticoid treatment of the frog Rana esculenta inhibits the transcription of all genes encoding antibacterial peptides by inducing the synthesis of IκBα. These results suggest that also in vertebrates peptide‐mediated innate immunity is controlled by NF‐κB‐regulated transcription.

Sequence of a gene from Bombina orientalis coding for the antimicrobial peptide BLP-7

The structure of a gene coding for bombinin-like peptides (BLP) in Bombina orientalis was determined. It comprises two exons separated by a 1337 bp intron. Exon 1 codes for the signal peptide, while exon 2 contains the genetic information for BLP-7 and a bombinin H-type peptide (GH-2). The promoter region contains putative recognition sites for nuclear factors, such as NF-IL6 and NF-kappaB. The analysis of the structure of this gene, compared with that of the previously reported BLP-3 gene sequence, suggests the occurrence of a gene duplication event, rather than an alternative splicing mechanism, which leads to the generation of both inter- and intra-families variability in this class of cytolytic peptides. Furthermore, chromosome walking analysis indicates that this gene family is not densely clustered.

Evidence that prokineticin receptor 2 exists as a dimer in vivo

Prokineticins are proteins that regulate diverse biological processes including gastrointestinal motility, angiogenesis, circadian rhythm, and innate immune response. Prokineticins bind two closed related G-protein coupled receptors (GPCRs), PKR1 and PKR2. In general, these receptors act as molecular switches to relay activation to heterotrimeric G-proteins and a growing body of evidence points to the fact that GPCRs exist as homo- or heterodimers. We show here by Western-blot analysis that PKR2 has a dimeric structure in neutrophils. By heterologous expression of PKR2 in Saccharomyces cerevisiae, we examined the mechanisms of intermolecular interaction of PKR2 dimerization. The potential involvement of three types of mechanisms was investigated: coiled-coil, disulfide bridges, and hydrophobic interactions between transmembrane domains. Characterization of differently deleted or site-directed PKR2 mutants suggests that dimerization proceeds through interactions between transmembrane domains. We demonstrate that co-expressing binding-deficient and signaling-deficient forms of PKR2 can re-establish receptor functionality, possibly through a domain-swapping mechanism.

A peptidylprolyl cis/trans isomerase from Xenopus laevis skin: cloning, biochemical characterization and putative role in the secretion

In amphibian skin secretions, a peptidylprolyl cis/trans isomerase activity was detected. A Xenopus laevis skin cDNA coding for this protein was cloned, sequenced and over-expressed in Escherichia coli. The primary structure of the protein shows extensive similarity with members of the cyclophilin A family. Catalytic parameters of the recombinant protein are similar to those of the human enzyme. The enzymatic activity is inhibited by cyclosporin A. Data suggesting that peptidylprolyl isomerization influences the biological activity of antibacterial peptides of amphibian origin are presented, and its putative role in the defence mechanism discussed.

Insight into the structure–function relationship of the nonheme iron halogenases involved in the biosynthesis of 4‐chlorothreonine – Thr3 from Streptomyces sp. OH‐5093 and SyrB2 from Pseudomonas syringae pv. syringae B301DR

Molecular cloning of the biosynthetic gene cluster involved in the production of free 4‐chlorothreonine in Streptomyces sp. OH‐5093 showed the presence of six ORFs: thr1, thr2, thr3, orf1, orf2 and thr4. According to bioinformatic analysis, thr1, thr2, thr3 and thr4 encode a free‐standing adenylation domain, a carrier protein, an Fe(II) nonheme α‐ketoglutarate‐dependent halogenase and a thioesterase, respectively, indicating the role of these genes in the activation and halogenation of threonine and the release of 4‐chlorothreonine in a pathway closely reflecting the formation of this amino acid in the biosynthesis of the lipodepsipeptide syringomycin from Pseudomonas syringae pv. syringae B301DR. Orf1 and orf2 show sequence similarity with alanyl/threonyl‐tRNA synthetases editing domains and drug metabolite transporters, respectively. We show that thr3 can replace the halogenase gene syrB2 in the biosynthesis of syringomycin, by functional complementation of the mutant P. s. pv. syringae strain BR135A1 inactivated in syrB2. We also provide an insight into the structure–function relationship of halogenases Thr3 and SyrB2 using homology modelling and site‐directed mutagenesis.

Expression of Bv8 in Pichia pastoris to identify structural features for receptor binding.

Bv8 is an amphibian peptide belonging to the widely distributed AVIT protein family. The mammalian orthologues of Bv8 were named prokineticin 1 and prokineticin 2. Two G-protein-coupled receptors for Bv8-prokineticins have been identified. The biological activities of Bv8/PK proteins range from angiogenesis and involvement in reproduction and cancer, to neuronal survival and neurogenesis, hypothalamic hormone secretion, circadian rhythm control and immunomodulatory processes. Identifying the structural determinants required for receptor binding of Bv8-PKs is mandatory for the design of PKR antagonists, which may be useful in the treatment and prevention of various disease states. Here we describe a procedure for the production in Pichia pastoris of Bv8 and 3 mutants: W24A-Bv8, in which the tryptophan in position 24 is substituted by alanine, the double mutant M1-W24A-Bv8, that contains an additional methionine at the N-terminus and Bv8-TyrTyr that includes two additional tyrosines at the C-terminus. The results evidence a relevant role of tryptophan 24 in Bv8-PKRs interaction.

Glutathione S-transferase, similar to sigma class, from skin secretions of Xenopus laevis

Using glutathione affinity chromatography followed by isoelectro‐focusing, we purified from the skin secretion of Xenopus laevis an isoenzyme of glutathione S‐transferase with an apparent subunit molecular mass of 22.5 kDa and an isoelectric point at pH 5.1. Its N‐terminal amino acid sequence was highly similar to that of the sigma class glutathione S‐transferase, which previously was demonstrated to have a glutathione‐dependent prostaglandin D2 synthase activity. Immunohistochemistry analysis revealed that the isoenzyme was located in the cytoplasm of granular gland cells.

The Bv8 gene from Bombina orientalis: Molecular cloning, genomic organization and functional characterization of the promoter

Bv8 is a secreted peptide from Bombina variegata skin glands with a molecular mass close to 8kDa that is conserved in fish, amphibians and mammals. Bv8 has diverse regulatory roles, including an involvement in hematopoiesis and immunomodulation. Here we report the genomic organization of the gene from Bombina orientalis coding for the Bv8 homolog (Bo8). It contains three exons separated by two large introns. Several putative transcription factor binding sites have been identified in the promoter sequence. Functional analysis of this region was performed using a yeast genetic system. The results indicate that the transcription factors AP-1, NF-kappaB and NFAT are involved in the regulation of the expression of Bo8. Hence, amphibians are a useful model for the study of transcriptional regulation of all Bv8 homologs.