Uncovering conserved patterns in bioactive peptides in Metazoa

Bioactive (neuro)peptides play critical roles in regulating most biological processes in animals. Peptides belonging to the same family are characterized by a typical sequence pattern that is conserved among the family's peptide members. Such a conserved pattern or motif usually corresponds to the functionally important part of the biologically active peptide. In this paper, all known bioactive (neuro)peptides annotated in Swiss-Prot and TrEMBL protein databases are collected, and the pattern searching program Pratt is used to search these unaligned peptide sequences for conserved patterns. The obtained patterns are then refined by combining the information on amino acids at important functional sites collected from the literature. All the identified patterns are further tested by scanning them against Swiss-Prot and TrEMBL protein databases. The diagnostic power of each pattern is validated by the fact that any annotated protein from Swiss-Prot and TrEMBL that contains one of the established patterns, is indeed a known (neuro)peptide precursor. We discovered 155 novel peptide patterns in addition to the 56 established ones in the PROSITE database. All the patterns cover 110 peptide families. Fifty-five of these families are not characterized by the PROSITE signatures, and 12 are also not identified by other existing motif databases, such as Pfam and SMART. Using the newly identified peptide signatures as a search tool, we predicted 95 hypothetical proteins as putative peptide precursors.     

MmtDB: a Metazoa mitochondrial DNA variants database.

The present paper describes the structure of MmtDB-a specialized database designed to collect Metazoa mitochondrial DNA variants. Priority in the data collection is given to the Metazoa species for which a large amount of variants is available, as it is the case for human variants. Starting from the sequences available in the Nucleotide Sequence Databases, the redundant sequences are removed and new sequences from other sources are added. Value-added information are associated to each variant sequence, e.g. analysed region, experimental method, tissue and cell lines, population data, sex, age, family code and information about the variation events (nucleotide position, involved gene, restriction site's gain or loss). Cross-references are introduced to the EMBL Data Library, as well as an internal cross-referencing among MmtDB entries according to their tissual, heteroplasmic, familiar and aplotypical correlation. MmtDB can be accessed through the World Wide Web at URL [see text].     

Update of MmtDB: a Metazoa mitochondrial DNA variants database.

The present paper describes the improvements in MmtDB, a specialised database designed to collect Metazoa mitochondrial DNA variants. Priority in the data collection has been given to Metazoa for which a large amount of variants is available, e.g., for humans. Starting from the sequences available in the Nucleotide Sequence Databases, the redundant sequences have been removed and new sequences from other sources have been added. Value-added information is associated to each variant sequence, e.g., analysed region, experimental method, tissue and cell lines, population data, sex, age, family code and information about the variation events (nucleotide position, involved gene, restriction site gain or loss). Cross-references are introduced to the EMBL Data Library, as well as an internal cross-referencing among MmtDB entries according to tissual, heteroplasmic, familiar and aplotypical correlation. Furthermore MmtDB has a new section, AMmtDB: Aligned Metazoan mitochondrial biosequences. MmtDB can be accessed through the World Wide Web at URL http://WWW.ba.cnr.it/[symbol: see text]areamt08/MmtDBWWW.htm     

Update of AMmtDB: a database of multi-aligned Metazoa mitochondrial DNA sequences

The AMmtDB database (http://bighost.area.ba.cnr.it/mitochondriome) has been updated by collecting the multi-aligned sequences of Chordata and Invertebrata mitochondrial genes coding for proteins and tRNAs. Links to the multi-aligned mtDNA intraspecies variants, collected in VarMmtDB at the Mitochondriome web site, have been introduced. The genes coding for proteins are multi-aligned based on the translated sequences and both the nucleotide and amino acid multi-alignments are provided. AMmtDB data selected through SRS can be viewed and managed using GeneDoc or other programs for the management of multi-aligned data depending on the user’s operative system. The multiple alignments have been produced with CLUSTALW and PILEUP programs and then carefully optimized manually.     

Development of affinity chromatography using a bioactive peptide as a ligand

By repeatedly introducing hydrophilic polyethylene glycol (PEG) spacer (2) onto affinity resin bearing a bioactive peptide (1/2 secretory leukocyte protease inhibitor, 1/2SLPI) as a ligand, the adsorption of nonspecific binding proteins was effectively reduced and the purification efficacy of elastase, which is one of the target molecules for 1/2SLPI, from a protein mixture was improved. Moreover, using this resin, we also successfully detected L-plastin, as an endogenous target molecule for SLPI, from HL-60 cell lysate.     

Telomere biology in Metazoa

In this review we present critical overview of some of the available literature on the fundamental biology of telomeres and telomerase in Metazoan. With the exception of Nematodes and Arthropods, the (TTAGGG)_n sequence is conserved in most Metazoa. Available data show that telomerase-based end maintenance is a very ancient mechanism in unicellular and multicellular organisms. In invertebrates, fish, amphibian, and reptiles persistent telomerase activity in somatic tissues might allow the maintenance of the extensive regenerative potentials of these species. Telomerase repression among birds and many mammals suggests that, as humans, they may use replicative aging as a tumor protection mechanism.     

The Role of Transposable Elements in Emergence of Metazoa

Systems initially emerged for protecting genomes against insertions of transposable elements and represented by mechanisms of splicing regulation, RNA–interference, and epigenetic factors have played a key role in the evolution of animals. Many studies have shown inherited transpositions of mobile elements in embryogenesis and preservation of their activities in certain tissues of adult organisms. It was supposed that on the emergence of Metazoa the self–regulation mechanisms of transposons related with the gene networks controlling their activity could be involved in intercellular cell coordination in the cascade of successive divisions with differentiated gene expression for generation of tissues and organs. It was supposed that during evolution species–specific features of transposons in the genomes of eukaryotes could form the basis for creation of dynamically related complexes of systems for epigenetic regulation of gene expression. These complexes could be produced due to the influence of noncoding transposon–derived RNAs on DNA methylation, histone modifications, and processing of alternative splicing variants, whereas the mobile elements themselves could be directly involved in the regulation of gene expression in cis and in trans. Transposons are widely distributed in the genomes of eukaryotes; therefore, their activation can change the expression of specific genes. In turn, this can play an important role in cell differentiation during ontogenesis. It is supposed that transposons can form a species–specific pattern for control of gene expression, and that some variants of this pattern can be favorable for adaptation. The presented data indicate the possible influence of transposons in karyotype formation. It is supposed that transposon localization relative to one another and to protein–coding genes can influence the species–specific epigenetic regulation of ontogenesis.     

Imaging the membrane lytic activity of bioactive peptide latarcin 2a

Latarcin 2a (ltc2a, GLFGKLIKKFGRKAISYAVKKARGKH-COOH) is a short linear antimicrobial and cytolytic peptide extracted from the venom of the Central Asian spider, Lachesana tarabaevi, with lytic activity against Gram-positive and Gram-negative bacteria, erythrocytes, and yeast at micromolar concentrations. Ltc2a adopts a helix-hinge-helix structure in membrane mimicking environment, whereas its derivative latarcin 2aG11A (ltc2aG11A, GLFGKLIKKFARKAISYAVKKARGKH-COOH), likely adopts a more rigid structure, demonstrates stronger nonspecific interaction with the zwitterionic membrane, and is potentially more toxic against eukaryotic cells. In this work, interactions of these two ltc2a derivatives with supported "raft" lipid bilayer (1,2-dioleoyl-sn-glycero-3-phosphocholin/egg sphingomyelin/cholesterol 40/40/20mol%) were studied by in situ atomic force microscopy in order to investigate the potential anticancer activity of the peptides since some breast and prostate cancer cell lines contain higher levels of cholesterol-rich lipid rafts than non-cancer cells. Both peptides induced reorganization of the raft model membrane by reducing line tension of the liquid ordered phase. Ltc2aG11A induced membrane thinning likely due to membrane interdigitation. Formation of large pores by the peptides in the bilayer was observed. Cholesterol was found to attenuate membrane disruption by the peptides. Finally, leakage assay showed that both peptides have similar membrane permeability toward various model membrane vesicles.     

Recombinant expression of bioactive peptide lunasin in Escherichia coli

Lunasin, a cancer-preventive peptide, was isolated from soybean, barley, and wheat. Previous studies showed that this 43-amino acid peptide has the ability to suppress chemical carcinogen-induced transformation in mammalian cells and skin carcinogenesis in mice. In this study, we attempted to use the Escherichia coli T7 expression system for expression of lunasin. The lunasin gene was synthesized by overlapping extension polymerase chain reaction and expressed in E. coli BL21(DE3) with the use of vector pET29a. The recombinant lunasin containing his-tag at the C-terminus was expressed in soluble form which could be purified by immobilized metal affinity chromatography. After 4 h, the expression level is above 4.73 mg of recombinant his-tagged lunasin/L of Luria–Bertani broth. It does not affect the bacterial growth and expression levels. This is the first study that successfully uses E. coli as a host to produce valuable bioactive lunasin. The result of in vitro bioassay showed that the purified recombinant lunasin can inhibit histone acetylation. Recombinant lunasin also inhibits the release of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and nitric oxide production). Compared with other research methods on extraction or chemical synthesis to produce lunasin, our method is very efficient in saving time and cost. In the future, it could be applied in medicine and structure–function determination.     

Experiment-specific estimation of peptide identification probabilities using a randomized database

Determining the error rate for peptide and protein identification accurately and reliably is necessary to enable evaluation and crosscomparisons of high throughput proteomics experiments. Currently, peptide identification is based either on preset scoring thresholds or on probabilistic models trained on datasets that are often dissimilar to experimental results. The false discovery rates (FDR) and peptide identification probabilities for these preset thresholds or models often vary greatly across different experimental treatments, organisms, or instruments used in specific experiments. To overcome these difficulties, randomized databases have been used to estimate the FDR. However, the cumulative FDR may include low probability identifications when there are a large number of peptide identifications and exclude high probability identifications when there are few. To overcome this logical inconsistency, this study expands the use of randomized databases to generate experiment-specific estimates of peptide identification probabilities. These experiment-specific probabilities are generated by logistic and Loess regression models of the peptide scores obtained from original and reshuffled database matches. These experiment-specific probabilities are shown to very well approximate "true" probabilities based on known standard protein mixtures across different experiments. Probabilities generated by the earlier Peptide_Prophet and more recent LIPS models are shown to differ significantly from this study's experiment-specific probabilities, especially for unknown samples. The experiment-specific probabilities reliably estimate the accuracy of peptide identifications and overcome potential logical inconsistencies of the cumulative FDR. This estimation method is demonstrated using a Sequest database search, LIPS model, and a reshuffled database. However, this approach is generally applicable to any search algorithm, peptide scoring, and statistical model when using a randomized database.     

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-β-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two l-glutamine residues. A substance P derivative with an N-acetyl-d-glucosamine residue attached to the fifth or sixth l-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-d-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the l-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the l-glutamine residue of the peptide was not liberated by peptide-N⁴-(N-acetyl-β-d-glucosaminyl) asparagine amidase F.     

Genetic manipulation of non-ribosomal peptide synthetases to generate novel bioactive peptide products

Non-ribosomal peptide synthetases (NRPS) are large modular enzymes that govern the synthesis of numerous biotechnologically relevant products. Their mode of action is frequently compared to an assembly line, in which each module acts in a semi-autonomous but coordinated manner to add a specific monomer to a growing peptide chain, unfettered by ribosomal constraints. The modular nature of these systems offers tantalising prospects for synthetic biology, wherein the assembly line is re-engineered at a genetic level to generate a specific or combinatorial modified product. However, despite some success stories, a “one size fits all” approach to NRPS synthetic biology remains elusive. This review examines both rational and random mutagenesis strategies that have been employed to modify NRPS function, in an attempt to highlight key points that should be considered when seeking to re-engineer an NRPS biosynthetic template.     

Effect of bioactive peptide on ram semen cryopreservation

This present study investigated the effect of bioactive peptide (BAPT) (BAPT) on the quality of ram semen during cryopreservation. Ram ejaculates were extended with Tris buffer supplemented with no antioxidants (as control group), 20 μg/mL BAPT (as BAPT20 group), 40 μg/mL BAPT (as BAPT40 group) and 60 μg/mL BAPT (as BAPT60 group). After cryopreservation, sperm quality including motility, vitality, the percentage of hypoosmotic swelling test (HOST)-positive spermatozoa and the percentage of intact acrosomes was assessed. Furthermore, the malondialdehyde (MDA) in seminal plasma and spermatozoa were analyzed, followed by the measurement of superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px) levels in seminal plasma. After in vitro fertilization, the embryonic cleavage rates and development rates of different groups were analyzed to compare the developmental abilities of spermatozoa. The results showed that the post-thaw sperm motility was significantly higher in the BAPT60 group compared to those in the BAPT20, BAPT40 and control groups (P < 0.05). The percentage of live sperms significantly increased from 48.12 ± 2.35% for the BAPT20 group, 55.43 ± 2.16% for the BAPT40 group to 57.53 ± 3.15% for the BAPT60 group. The percentage of HOST-positive spermatozoa was significantly higher in the BAPT60 group than those in BAPT20, BAPT40 and control groups (P < 0.05). The MDA levels in seminal plasma and spermatozoa were significantly reduced with BAPT supplement (P < 0.05). Additionally, the SOD, CAT and GSH-Px levels in the BAPT experimental groups were significantly higher than those of the control group, which further indicated that BAPT significantly inhibit the reactive oxygen species (ROS) production during the cryopreservation of ram semen. Furthermore, the embryonic cleavage rates and development rates of the BAPT40 and BAPT60 groups were significantly increased in comparison with the BAPT20 and control groups (P < 0.05).In conclusion, BAPT improved the ram sperm quality via inhibiting the ROS production during cryopreservation, and could be applied as a promising supplement for ram semen cryopreservation.     

Detecting bioactive amyloid beta peptide species in Alzheimer's disease

Amyloid beta peptide (A beta) is believed to play a central role in the pathogenesis of Alzheimer's disease (AD). However, the form of A beta that induces neurodegeneration in AD, defined here as bioactive A beta, is not clear. Preventing the formation of bioactive A beta or inactivating previously formed bioactive A beta should be a promising approach to treat AD. We have previously developed a cell-based assay for the detection of bioactive A beta species. The assay is based upon the correlation between the ability of an A beta sample to induce a unique form of cellular MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] formazan exocytosis, and its ability to activate glia and induce neurotoxicity. Here, we show that this cell-based assay is not only useful for a cellular model of A beta amyloidogenesis but is also able to detect bioactive A beta species in a transgenic mouse model of AD, as well as in post-mortem cortex samples from AD patients. There is a good correlation between the extent of glia activation and the level of bioactive A beta species in the mouse brain. A promising deuteroporphyrin that can inactivate bioactive A beta species was also identified using this assay. These novel insights and findings should have important implications for the treatment of AD.     

Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide

Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52 ± 2.44E-10 and 5.87 ± 1.3E–9 M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33 ± 1.15E-9 and 4.11 ± 1.09E–9 M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P < 0.05). They also significantly reduced the serum sodium level and increased the urine volume (P < 0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity.     

Development of genetic markers in abalone through construction of a SNP database

In the absence of a reference genome, single-nucleotide polymorphisms (SNP) discovery in a group of abalone species was undertaken by random sequence assembly. A web-based interface was constructed, and 11 932 DNA sequences from the genus Haliotis were assembled, with 1321 contigs built. Of these, 118 contigs that consisted of at least ten annotation groups were selected. The 1577 putative SNPs were identified from the 118 contigs, with SNPs in several HSP70 gene contigs confirmed by PCR amplification of an 809-bp DNA fragment. SNPs in the HSP70 gene were compared across eight abalone species. A total of 129 polymorphic sites, including heterozygote sites within and among species, were observed. Phylogenetic analysis of the partial HSP70 gene region showed separation of the tested abalone into two groups, one reflecting the southern hemisphere species and the other the northern hemisphere species. Interestingly, Haliotis iris from New Zealand showed a closer relationship to species distributed in the northern Pacific region. Although HSP genes are known to be highly conserved among taxa, the validation of polymorphic SNPs from HSP70 in this mollusc demonstrates the applicability of cross-species SNP markers in abalone and the first step towards universal nuclear markers in Haliotis.     

JenPep: a database of quantitative functional peptide data for immunology

MOTIVATION: The compilation of quantitative binding data underlies attempts to derive tools for the accurate prediction of epitopes in cellular immunology and is part of our concerted goal to develop practical computational vaccinology. RESULTS: JenPep is a family of relational databases supporting the growing community of immunoinformaticians. It contains quantitative data on peptide binding to Major Histocompatibility Complexes (MHCs) and to Transmembrane Peptide Transporter (TAP), as well as an annotated list of T-cell epitopes. AVAILABILITY: The database is available via the Internet. An HTML interface allowing searching of the database can be found at the following address: http://www.jenner.ac.uk/JenPep.