Transcriptome-based analysis reveals that the biosynthesis of anthocyanins is more active than that of flavonols and proanthocyanins in the colorful flowers of Lagerstroemia indica.

Mechanisms associated with the control of flower color in crape myrtle varieties have yet to be sufficiently elucidated, which has tended to hamper the use of modern molecular and genetic strategies in the breeding programs for this plant. The whole transcriptome of four L. indica varieties characterized by different flower colors (white, light purple, deep purplish pink, and strong red) was sequenced, and we performed bioinformatic, quantitative PCR, and co-expression analyses of R2R3 MYB transcription factor and anthocyanin/flavonol pathway genes. We obtained a total of 49,980 transcripts with full-length coding sequences. Both transcriptome and qPCR analyses revealed that anthocyanin/flavonol pathway genes were differentially expressed among the four different flowers types, with the expression of LiPAL, LiCHS, LiCHI, LiDFR, LiANS/LDOX, and LiUFGT being induced in colorful flowers, whereas that of LiF3´5´H, LiFLS, and LiLAR was found to be inhibited. Base on phylogenetic analysis, seven R2R3 MYB transcriptional factors were identified as putative regulators of flower color. The molecular characteristics and co-expression patterns indicated that these MYBs differentially modulate their target genes, with two probably acting as activators, three as repressors, and one contributing to the regulation of vacuolar pH. The findings of this study indicate that the anthocyanin biosynthesis is more active than the flavonol and proanthocyanin in the colorful flowers. These observations provide new genomic information on L. indica and contribute gene resources for the flower color-targeted breeding of crape myrtle.

Reconstitution and Substrate Specificity of the Thioether-Forming Radical S-Adenosylmethionine Enzyme in Freyrasin Biosynthesis.

The radical non-α-carbon thioether peptides (ranthipeptides) are a newly described class of ribosomally synthesized and post-translationally modified peptide (RiPP). Ranthipeptide biosynthetic gene clusters are characterized by a Cys-rich precursor peptide and a radical S-adenosylmethionine (rSAM)-dependent enzyme that forms a thioether linkage between a Cys donor and an acceptor residue. Unlike the sulfur-to-α-carbon linked thioether peptides (sactipeptides), known ranthipeptides contain thioethers to either the ß- or γ-carbon (i.e., non-α-carbon) of an acceptor residue. Recently, we reported the discovery of freyrasin, a ranthipeptide from Paenibacillus polymyxa, which contains six thioethers from Cys-X3-Asp motifs present in the precursor peptide (PapA). The linkages are exclusively to the ß-carbon of Asp (S-Cß). In this report, we performed mutational analysis of PapA and the cognate thioether-forming rSAM enzyme (PapB) to define the substrate scope. Using a mass spectrometry-based activity assay, our data show that PapB is intolerant toward Ala and Asn in the acceptor position but tolerates Glu-containing variants. NMR spectroscopic data of a Glu variant demonstrated that the thioether linkage was to the 4-position of Glu (S-Cγ). Furthermore, we demonstrate that PapB is intolerant to expansion and contraction of the thioether motifs (Cys-Xn-Asp, n = 2 or 4), although a minimal substrate featuring only one Cys-X3-Asp motif was competent for thioether formation. Akin to the sactipeptides, PapB was dependent on a RiPP recognition element (RRE) to bind the cognate precursor peptide, with deletion resulting in loss-of-function in vivo. The activity of PapB could be restored in vivo by supplying the excised RRE in trans. Finally, we reconstituted the activity of PapB in vitro, which led to modification of all six Cys residues in PapA. These studies provide insights into ranthipeptide biosynthesis and expand our understanding of rSAM enzyme chemistry in natural product biosynthesis.

The Prebiotic C-Terminal Elongation of Peptides Can Be Initiated by N-Carbamoyl Amino Acids.

The formation of peptides upon 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-promoted activation of N-carbamoylamino acids (CAA), was considered in the scope of our recent works on carbodiimide promoted C-terminus elongation of peptides in a prebiotic context. Thus EDC promoted activation of CAA derivatives of Tyr(Me) or Ala in dilute aqueous medium pH 5.5-6.5 in the presence of excess of AA, resulted in peptide formation by C-terminus activation/elongation. Kinetic results similar to those of EDC-mediated activation of N-acyl-AA lead us to postulate the formation of a 2-amino-5(4H)-oxazolone intermediate by cyclization of the activated CAA, in spite of the absence of epimerization occurred at CAA residues. Thus, in a prebiotic context, CAA may have played a similar role as N-acyl-AA in the initiation of C-terminus peptide elongation.

Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif.

A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications. However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a "difficult peptide". In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification. The maximum expression level was observed when the peptide was fused to thioredoxin and the optimized purification process consisted of three chromatographic steps: immobilized-metal affinity chromatography followed by cation-exchange chromatography and, finally, a reverse-phase high-performance liquid chromatography. This entire process led to a final volumetric production yield of 1.5 mg of pure Kcoil peptide per liter of bacterial culture, which represents a significant step towards the cost-effective production and application of coiled-coil motifs. Our results thus demonstrate for the first time that bacterial production is a viable alternative to the chemical synthesis of de novo designed coil peptides.

Elastin-like polypeptide based nanoparticles: design rationale toward nanomedicine.

Elastin-like polypeptides (ELPs) are characterized by a high sequence control, temperature responsiveness and biocompatibility, which make them highly interesting as smart materials for application in nanomedicine. In particular the construction of ELP-based nanoparticles has recently become a focal point of attention in materials research. This review will give an overview of the ELP-based nanoparticles that have been developed until now and their underlying design principles. First a short introduction on ELPs and their stimulus-responsive behavior will be given. This characteristic has been applied for the development of ELP-based block copolymers that can self-assemble into nanoparticles. Both the fully ELP-based as well as several ELP hybrid materials that have been reported to form nanoparticles will be discussed, which is followed by a concise description of the promising biomedical applications reported for this class of materials.

Parâmetros salivares, proteoma e saúde bucal na síndrome de Moebius / Salivary parameters, proteome and oral health in Moebios syndrome

A síndrome de Moebius (SM) é uma diplegia congênita rara caracterizada por paralisia total ou parcial do VI e VII nervos cranianos, que leva à ausência ou deficiência dos movimentos dos músculos envolvidos na mímica facial e ao estrabismo convergente. As características bucais descritas nesses indivíduos incluem o palato ogival, micrognatia, malformação de língua, filtro curto, falta de coaptação de lábios, e maior incidência de lesões de cárie. O objetivo deste estudo foi avaliar as características salivares quantitativas e qualitativas, incluindo o proteoma salivar, de indivíduos com SM, associá-las com a saúde bucal, e compará-las com as características salivares de um grupo controle, não afetado pela SM. Foram incluídos 15 indivíduos com SM e 15 controles. O comprometimento facial do individuos com a SM foi avaliada e graduada em scores 0,1 ou 2, uni ou bilateral, para os nervos II, III, IV, V, VI, VII e XI. Os pesquisadores determinaram o índice de cárie (ICDAS), de doença periodontal (PSR) e de placa (Silness Lõe) nos dois grupos de estudo. Também realizaram coletas de saliva total não estimulada, estimulada e parotídea bilateral, sendo o fluxo salivar estabelecido em ml/min. A capacidade tampão foi avaliada na saliva total estimulada através da titulação de HCl 0,01N. A atividade de ?-amilase nas amostrasmfoi medida através da produção de maltose. Para a análise proteômica optou-se pela divisão das amostras de saliva de acordo com o fluxo em ml/min. Desta forma, para cada grupo, estudo e controle, os 4 tipos de saliva (estimulada, não estimulada, parotídea esquerda, parotídea direita) foram subdivididos de acordo com baixo fluxo (abaixo da média do grupo) ou alto fluxo (acima da média do grupo), resultando em 16 subgrupos.

O proteoma foi obtido por duas metodologias distintas, a primeira a partir da cromatografia líquida espectrometria de massas e a segunda que utilizou a técnica de eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE) associada à eletroforese em gel de poliacrilamida (native cationic). A ocorrência das lesões de cárie foi significativamente mais alta entre os participantes com SM (p>0,05) no corte 2, bem como a ocorrência de doença periodontal (p>0,05), quando comparado ao grupo controle. Não houve diferença no índice de placa entre os grupos. A análise proteômica mostrou diminuição de cistatinas B, S e SN nos indivíduos com SM. Não houve diferença no perfil proteico entre os grupos de baixo e de alto fluxo salivar, para indivíduos com SM e controle. Houve aumento na quantidade de amilase salivar em e de histatina 1,3 e 5 em indivíduos com SM. Concluímos que indivíduos com SM apresentam diminuição de fluxo salivar, de capacidade tampão e alterações proteicas que colocam esses indivíduos em situação de maior risco para cárie e para doença periodontal.

The Moebius syndrome (MS) is a rare congenital diplegia characterized by total or partial palsy of the VI and VII cranial nerves, leading to the absence or disability of the movements of facial expression muscles and to convergent strabismus. The oral features described in these individuals include high-arched palate, micrognathia, tongue malformation, short filter, lack of lips coaptation, and higher incidence of caries lesions. The aim of this study was to evaluate the quantitative and qualitative salivary characteristics, including the salivary proteome of individuals with MS, associate them with the oral health, and compare them to the salivary characteristics of a control group, unaffected by SM. We included 15 subjects with MS and 15 controls. The facial involvement of individuals with MS was evaluated and graded on scores 0, 1 or 2, uni or bilateral to the nerves II, III, IV, V, VI, VII and XI. The researchers established the caries (ICDAS), periodontal disease (PSR) and plate (Silness Lõe) indexes in both groups. We also performed unstimulated, stimulated and bilateral parotid saliva collections, and salivary flow was calculated (ml / min). The buffer capacity was measured in stimulated saliva by titration of 0.01N HCl. The ?-amylase activity was determined by maltose production. For proteomic analysis it was decided to split the saliva samples in accordance with the flow in ml/min. Thus, for each group, study and control, the 4 types of saliva (stimulated, unstimulated, left parotid, right parotid) were subdivided according to low flow (below the group average) or high flow (above average group), resulting in 16 subgroups.

The proteome was obtained by two different methodologies, the first was liquid chromatography mass spectrometry and the second was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) associated with cationic PAGE. The occurrence of caries lesions, related to cut-off 2, as well as the occurrence of periodontal disease, was significantly higher (p> 0.05) in participants with MS when compared to the control group. There was no statistical difference in plaque index between groups. Proteomics analysis showed decrease of cystatin B, S and SN in individuals with MS. There was no difference in protein profile between the low and high salivary flow groups, for individuals with MS and control. There was an increase in the amylase amount and histatin 1, 3 and 5 in individuals with MS. We concluded that individuals with MS present decreased salivary flow, decreased buffer capacity and protein alterations that place these individuals at increased risk for caries and periodontal disease.

Parâmetros salivares, proteoma e saúde bucal na síndrome de Moebius / Salivary parameters, proteome and oral health in Moebios syndrome

A síndrome de Moebius (SM) é uma diplegia congênita rara caracterizada por paralisia total ou parcial do VI e VII nervos cranianos, que leva à ausência ou deficiência dos movimentos dos músculos envolvidos na mímica facial e ao estrabismo convergente. As características bucais descritas nesses indivíduos incluem o palato ogival, micrognatia, malformação de língua, filtro curto, falta de coaptação de lábios, e maior incidência de lesões de cárie. O objetivo deste estudo foi avaliar as características salivares quantitativas e qualitativas, incluindo o proteoma salivar, de indivíduos com SM, associá-las com a saúde bucal, e compará-las com as características salivares de um grupo controle, não afetado pela SM. Foram incluídos 15 indivíduos com SM e 15 controles. O comprometimento facial do individuos com a SM foi avaliada e graduada em scores 0,1 ou 2, uni ou bilateral, para os nervos II, III, IV, V, VI, VII e XI. Os pesquisadores determinaram o índice de cárie (ICDAS), de doença periodontal (PSR) e de placa (Silness Lõe) nos dois grupos de estudo. Também realizaram coletas de saliva total não estimulada, estimulada e parotídea bilateral, sendo o fluxo salivar estabelecido em ml/min. A capacidade tampão foi avaliada na saliva total estimulada através da titulação de HCl 0,01N. A atividade de ?-amilase nas amostrasmfoi medida através da produção de maltose. Para a análise proteômica optou-se pela divisão das amostras de saliva de acordo com o fluxo em ml/min. Desta forma, para cada grupo, estudo e controle, os 4 tipos de saliva (estimulada, não estimulada, parotídea esquerda, parotídea direita) foram subdivididos de acordo com baixo fluxo (abaixo da média do grupo) ou alto fluxo (acima da média do grupo), resultando em 16 subgrupos. O proteoma foi obtido por duas metodologias distintas, a primeira a partir da cromatografia líquida espectrometria de massas e a segunda que utilizou a técnica de eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE) associada à eletroforese em gel de poliacrilamida (native cationic). A ocorrência das lesões de cárie foi significativamente mais alta entre os participantes com SM (p>0,05) no corte 2, bem como a ocorrência de doença periodontal (p>0,05), quando comparado ao grupo controle. Não houve diferença no índice de placa entre os grupos. A análise proteômica mostrou diminuição de cistatinas B, S e SN nos indivíduos com SM. Não houve diferença no perfil proteico entre os grupos de baixo e de alto fluxo salivar, para indivíduos com SM e controle. Houve aumento na quantidade de amilase salivar em e de histatina 1,3 e 5 em indivíduos com SM. Concluímos que indivíduos com SM apresentam diminuição de fluxo salivar, de capacidade tampão e alterações proteicas que colocam esses indivíduos em situação de maior risco para cárie e para doença periodontal.

The Moebius syndrome (MS) is a rare congenital diplegia characterized by total or partial palsy of the VI and VII cranial nerves, leading to the absence or disability of the movements of facial expression muscles and to convergent strabismus. The oral features described in these individuals include high-arched palate, micrognathia, tongue malformation, short filter, lack of lips coaptation, and higher incidence of caries lesions. The aim of this study was to evaluate the quantitative and qualitative salivary characteristics, including the salivary proteome of individuals with MS, associate them with the oral health, and compare them to the salivary characteristics of a control group, unaffected by SM. We included 15 subjects with MS and 15 controls. The facial involvement of individuals with MS was evaluated and graded on scores 0, 1 or 2, uni or bilateral to the nerves II, III, IV, V, VI, VII and XI. The researchers established the caries (ICDAS), periodontal disease (PSR) and plate (Silness Lõe) indexes in both groups. We also performed unstimulated, stimulated and bilateral parotid saliva collections, and salivary flow was calculated (ml / min). The buffer capacity was measured in stimulated saliva by titration of 0.01N HCl. The ?-amylase activity was determined by maltose production. For proteomic analysis it was decided to split the saliva samples in accordance with the flow in ml/min. Thus, for each group, study and control, the 4 types of saliva (stimulated, unstimulated, left parotid, right parotid) were subdivided according to low flow (below the group average) or high flow (above average group), resulting in 16 subgroups. The proteome was obtained by two different methodologies, the first was liquid chromatography mass spectrometry and the second was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) associated with cationic PAGE. The occurrence of caries lesions, related to cut-off 2, as well as the occurrence of periodontal disease, was significantly higher (p> 0.05) in participants with MS when compared to the control group. There was no statistical difference in plaque index between groups. Proteomics analysis showed decrease of cystatin B, S and SN in individuals with MS. There was no difference in protein profile between the low and high salivary flow groups, for individuals with MS and control. There was an increase in the amylase amount and histatin 1, 3 and 5 in individuals with MS. We concluded that individuals with MS present decreased salivary flow, decreased buffer capacity and protein alterations that place these individuals at increased risk for caries and periodontal disease.

Combination of theoretical and experimental approaches for the design and study of fibril-forming peptides.

Self-assembling peptides that can form supramolecular structures such as fibrils, ribbons, and nanotubes are of particular interest to modern bionanotechnology and materials science. Their ability to form biocompatible nanostructures under mild conditions through non-covalent interactions offers a big biofabrication advantage. Structural motifs extracted from natural proteins are an important source of inspiration for the rational design of such peptides. Examples include designer self-assembling peptides that correspond to natural coiled-coil motifs, amyloid-forming proteins, and natural fibrous proteins. In this chapter, we focus on the exploitation of structural information from beta-structured natural fibers. We review a case study of short peptides that correspond to sequences from the adenovirus fiber shaft. We describe both theoretical methods for the study of their self-assembly potential and basic experimental protocols for the assessment of fibril-forming assembly.

Peptide formation by N-methyl amino acids in translation is hastened by higher pH and tRNA(Pro).

Applications of N-methyl amino acids (NMAAs) in drug discovery are limited by their low efficiencies of ribosomal incorporation, and little is known mechanistically about the steps leading to incorporation. Here, we demonstrate that a synthetic tRNA body based on a natural N-alkyl amino acid carrier, tRNA(Pro), increases translation incorporation rates of all three studied NMAAs compared with tRNA(Phe)- and tRNA(Ala)-based bodies. We also investigate the pH dependence of the incorporation rates and find that the rates increase dramatically in the range of pH 7 to 8.5 with the titration of a single proton. Results support a rate-limiting peptidyl transfer step dependent on deprotonation of the N-nucleophile of the NMAA. Competition experiments demonstrate that several futile cycles of delivery and rejection of A-site NMAA-tRNA are required per peptide bond formed and that increasing magnesium ion concentration increases incorporation yield. Data clarify the mechanism of ribosomal NMAA incorporation and provide three generalizable ways to improve incorporation of NMAAs in translation.

Recombinant human elastin-like magnetic microparticles for drug delivery and targeting.

Bioinspired recombinant polypeptides represent a highly promising tool in biomedical research, being protein intrinsic constituents of both cells and their natural matrices. In this regard, a very interesting model is represented by polypeptides inspired by elastin, which naturally confers rubber-like elasticity to tissues, and is able to undergo wide deformations without rupture. In this paper, a microparticle system based on a recombinant human elastin-like polypeptide (HELP) is reported for drug delivery applications. HELP microparticles are prepared through a water-in-oil emulsion of an aqueous solution of recombinant polypeptide in isoctane, followed by enzymatic cross-linking. Superparamagnetic iron oxide nanoparticles are introduced in this system with the purpose of conferring magnetic properties to the microspheres, and thus controlling their targeting and tracking as drug vectors. The obtained microparticles are characterized in terms of morphology, structure, magnetic properties, drug release, and magnetic drivability, showing interesting and promising results for further biomedical applications.

Increase in antioxidant and antihypertensive peptides from Argentinean wines by Oenococcus oeni.

Cells from an exponential Oenococcus oeni m1 culture in a grape juice medium were inoculated into a synthetic wine medium (SW) supplemented with a protein and polypeptide fraction (PPF) of high molecular weight (higher than 12,400 Da) obtained from four varietals of Cafayate Argentinean wines. O. oeni maintains viability after 48 h incubation time and enables the increase in extracellular proteolytic activity and the release of low molecular weight peptides by 1.067, 0.397, 0.915 and 0.705 mg N/L in the respective SW supplemented with PPF from Cabernet Sauvignon, Malbec, Tannat and Torrontés wine varietals. After 48 h incubation time, concomitantly with peptide release, an increase in antioxidant and antihypertensive activities was detected in all studied media. The highest increase was detected in the presence of PPF from Cabernet and Tannat wine varietals. Maximum increase in antioxidant activity (366.1 µmol FeSO4/L in the case of ferric reducing antioxidant power and 8.9% in 2,2-diphenyl-1-picrylhydrazyl radical scavenging) was produced by the peptides released from PPF of Cabernet Sauvignon wine. The peptides released from PPF Tannat wine varietal caused the highest increase in antihypertensive activity (56.2% in angiotensin I-converting enzyme inhibitory activity). Oenococcus oeni m1 would provide additional benefits to wine such as an increase in bioactive peptides with multifunctional beneficial activities.

A model for the role of isomerization in nascent peptide movement through the ribosomal tunnel.

The ribosome is a molecular machine whose manner of controlling the progression of the nascent chain through the ribosomal exit tunnel is currently unknown. A novel model for the mechanism driving the nascent chain motion is hereby presented, in which the ∼180° rotatory motion performed by each C-terminal amino acid of the nascent chain during its translocation from the A site to the P site, is suggested to twist the newly formed peptide bond into cis conformation. By catalyzing the cis to trans isomerization, the ribosome is proposed to release the potential energy stored within the cis conformer and to utilize it to push the chain down the tunnel, thus operating as a molecular motor. This hypothetical isomerization mechanism is supported by its ability to provide an explanation for the peculiar conduct observed in translational events of nascent chains with C-terminal prolines: the slow peptide bond formation with puromycin, translation arrest, and tmRNA tagging.

Péptidos bioactivos / Bioactive peptides

Los péptidos bioactivos o péptidos con actividad biológica producidos durante la digestión gastrointestinal o la elaboración de alimentos pueden ejercer un importante papel en la regulación y la modulación metabólica, que sugiere su uso potencial como nutracéuticos e ingredientes de alimentos funcionales para promoción de la salud y la reducción del riesgo de enfermedad. En los últimos años se han destinado muchos esfuerzos al estudio de las diferentes actividades beneficiosas que estos péptidos bioactivos pueden tener sobre el organismo, incluyendo su actividad antihipertensiva, hipocolesterolemiante, antioxidante, antimicrobianae inmunomoduladora, así como su efecto opiáceo. Así mismo se están destinando esfuerzos en investigación para la detección de fuentes alimentarias de péptidos bioactivos así como al estudio de su biodisponibilidad, de sus propiedades funcionales y de sus mecanismos de acción (AU)

Bioactive peptides, or peptides with biological activity produced during gastrointestinal digestion or food processing, could play an important role in metabolic regulation and modulation, suggesting their potential use as nutraceuticals and ingredients of functional foods to promote health and reduce the risk of disease. In the last few years, efforts have been made to study the various potential beneficial activities of these bioactive peptides in the body, including their antihypertensive, hypocholesterolemic, antioxidant, antimicrobial, immunomodulatory, and opiate-like activities. Likewise, research is currently being carried out to detect food sources of bioactive peptides as well as to study their bioavailability, functional properties and mechanisms of action (AU)

Neuropathy-induced spinal GAP-43 expression is not a main player in the onset of mechanical pain hypersensitivity.

Structural plasticity within the spinal nociceptive network may be fundamental to the chronic nature of neuropathic pain. In the present study, the spatiotemporal expression of growth-associated protein-43 (GAP-43), a protein which has been traditionally implicated in nerve fiber growth and sprouting, was investigated in relation to mechanical pain hypersensitivity. An L5 spinal nerve transection model was validated by the presence of mechanical pain hypersensitivity and an increase in the early neuronal activation marker cFos within the superficial spinal dorsal horn upon innocuous hindpaw stimulation. Spinal GAP-43 was found to be upregulated in the superficial L5 dorsal horn from 5 up to 10 days after injury. GAP-43 was co-localized with calcitonin-gene related peptide (CGRP), but not vesicular glutamate transporter-1 (VGLUT-1), IB4, or protein kinase-γ (PKC-γ), suggesting the regulation of GAP-43 in peptidergic nociceptive afferents. These GAP-43/CGRP fibers may be indicative of sprouting peptidergic fibers. Fiber sprouting largely depends on growth factors, which are typically associated with neuro-inflammatory processes. The putative role of neuropathy-induced GAP-43 expression in the development of mechanical pain hypersensitivity was investigated using the immune modulator propentofylline. Propentofylline treatment strongly attenuated the development of mechanical pain hypersensitivity and glial responses to nerve injury as measured by microglial and astroglial markers, but did not affect neuropathy-induced levels of spinal GAP-43 or GAP-43 regulation in CGRP fibers. We conclude that nerve injury induces structural plasticity in fibers expressing CGRP, which is regarded as a main player in central sensitization. Our data do not, however, support a major role of these structural changes in the onset of mechanical pain hypersensitivity.

Two classes of new peptaibols are synthesized by a single non-ribosomal peptide synthetase of Trichoderma virens.

Peptaibols are a group of small peptides having a high α-aminoisobutyric acid (Aib) content and produced by filamentous fungi, especially by the members of the genus Trichoderma (anamorph Hypocrea). These antibiotics are economically important for their anti-microbial and anti-cancer properties as well as ability to induce systemic resistance in plants against microbial invasion. In this study we present sequences of two classes (11-residue and 14-residue) of peptaibols produced by the biocontrol fungus Trichoderma virens. Of the 35 11-residue peptaibols sequenced, 18 are hitherto not described, and all the 53 14-residue sequences described by us here are new. We have also identified a peptaibol synthetase (non-ribosomal peptide synthetase, NRPS) with 14 complete modules in the genome of this fungus and disruption of this single gene (designated as tex2) resulted in the loss of both the classes of peptaibols. We, thus present here an unprecedented case where a single NRPS encodes for two classes of peptaibols. The new peptaibols identified here could have applications as therapeutic agents for the management of human and plant health.

Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis.

BACKGROUND: Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS) in non-ribosomal peptide ligation. RESULTS: Here we describe novel examples of AAtRS related proteins that are likely to be involved in the synthesis of widely distributed peptide-derived metabolites. Using sensitive sequence profile methods we show that the cyclodipeptide synthases (CDPSs) are members of the HUP class of Rossmannoid domains and are likely to be highly derived versions of the class-I AAtRS catalytic domains. We also identify the first eukaryotic CDPSs in fungi and in animals; they might be involved in immune response in the latter organisms. We also identify a paralogous version of the methionyl-tRNA synthetase, which is widespread in bacteria, and present evidence using contextual information that it might function independently of protein synthesis as a peptide ligase in the formation of a peptide- derived secondary metabolite. This metabolite is likely to be heavily modified through multiple reactions catalyzed by a metal-binding cupin domain and a lysine N6 monooxygenase that are strictly associated with this paralogous methionyl-tRNA synthetase (MtRS). We further identify an analogous system wherein the MtRS has been replaced by more typical peptide ligases with the ATP-grasp or modular condensation-domains. CONCLUSIONS: The prevalence of these predicted biosynthetic pathways in phylogenetically distant, pathogenic or symbiotic bacteria suggests that metabolites synthesized by them might participate in interactions with the host. More generally, these findings point to a complete spectrum of recruitment of AAtRS to various non-ribosomal biosynthetic pathways, ranging from the conventional AAtRS, through closely related paralogous AAtRS dedicated to certain pathways, to highly derived versions of the class-I AAtRS catalytic domain like the CDPSs. Both the conventional AAtRS and their closely related paralogs often provide aminoacylated tRNAs for peptide ligations by MprF/Fem/MurM-type acetyltransferase fold ligases in the synthesis of peptidoglycan, N-end rule modifications of proteins, lipid aminoacylation or biosynthesis of antibiotics, such as valinamycin. Alternatively they might supply aminoacylated tRNAs for other biosynthetic pathways like that for tetrapyrrole or directly function as peptide ligases as in the case of mycothiol and those identified here.

Prolyl aminopeptidase from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 and synthesis of proline-containing peptides by its S144C variant.

We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 (PAP14270) was obtained using sequence-based screening. From PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.

Desiccation-induced structuralization and glass formation of group 3 late embryogenesis abundant protein model peptides.

Anhydrobiotic (i.e., life without water) organisms are known to produce group 3 late embryogenesis abundant (G3LEA) proteins during adaptation to severely water-deficient conditions. Their primary amino acid sequences are composed largely of loosely conserved 11-mer repeat units. However, little information has been obtained for the structural and functional roles of these repeat units. In this study, we first explore the consensus sequences of the 11-mer repeat units for several native G3LEA proteins originating from anhydrobiotic organisms among insects (Polypedilum vanderplanki), nematodes, and plants. Next, we synthesize four kinds of model peptides (LEA models), each of which consists of four or two repeats of the 11-mer consensus sequences for each of the three organisms. The structural and thermodynamic properties of the LEA models were examined in solution, in dehydrated and rehydrated states, and furthermore in the presence of trehalose, since a great quantity of this sugar is known to be produced in the dried cells of most anhydrobiotic organisms. The results of Fourier transform infrared (FTIR) spectroscopic measurements indicate that all of the LEA models transform from random coils to alpha-helical coiled coils on dehydration and return to random coils again on rehydration, both with and without trehalose. In contrast, such structural changes were never observed for a control peptide with a randomized amino acid sequence. Furthermore, our differential scanning calorimetry (DSC) measurements provide the first evidence that the above 11-mer motif-containing peptides themselves vitrify with a high glass transition temperature (>100 degrees C) and a low enthalpy relaxation rate. In addition, they play a role in reinforcing the glassy matrix of the coexisting trehalose. On the basis of these results, we discuss the underlying mechanism of G3LEA proteins as desiccation stress protectants.

Antimicrobial peptides present in mammalian skin and gut are multifunctional defence molecules.

Antimicrobial peptides are major components of the innate immune defence. They are well conserved along evolution, non-toxic and they ensure potent defences against a large number of pathogens. They act by direct killing of microorganisms and they possess additional roles in the regulation of adaptive immune responses, by recruting or stimulating immune cells. Skin and gut are positioned at the interface of internal milieu and external environment. They represent a physical and chemical barrier against pathogens invasion and the antimicrobial peptides limit pathogen growth in normal conditions. During infection or injury, some of these peptides are overexpressed and disrupt microbial membranes and/or stimulate immune cell recruitment, allowing to return to homeostasis or to increase inflammation. Antimicrobial peptides expression is altered in several diseases: alpha-defensins deficiency is related with Crohn's disease and in skin, cathelicidin LL-37 and beta-defensin-2 are overexpressed in psoriasis, while in atopic dermatitis, their expression is decreased. The present review provides an up-to-date summary of the expression and the biological roles of the antimicrobial peptides found in the skin and gastrointestinal mucosa of the host, in normal and pathological conditions. The involvement of these natural antimicrobial peptides in inflammation, is also discussed.

Chapter 8. Methods for in silico prediction of microbial polyketide and nonribosomal peptide biosynthetic pathways from DNA sequence data.

Fore-knowledge of the secondary metabolic potential of cultivated and previously uncultivated microorganisms can potentially facilitate the process of natural product discovery. By combining sequence-based knowledge with biochemical precedent, translated gene sequence data can be used to rapidly derive structural elements encoded by secondary metabolic gene clusters from microorganisms. These structural elements provide an estimate of the secondary metabolic potential of a given organism and a starting point for identification of potential lead compounds in isolation/structure elucidation campaigns. The accuracy of these predictions for a given translated gene sequence depends on the biochemistry of the metabolite class, similarity to known metabolite gene clusters, and depth of knowledge concerning its biosynthetic machinery. This chapter introduces methods for prediction of structural elements for two well-studied classes: modular polyketides and nonribosomally encoded peptides. A bioinformatics tool is presented for rapid preliminary analysis of these modular systems, and prototypical methods for converting these analyses into substructural elements are described.