Amino Acid Sequence Of Peptide Research Articles

Brevinin-2GHk from Sylvirana guentheri and the Design of Truncated Analogs Exhibiting the Enhancement of Antimicrobial Activity.

Brevinins are an important antimicrobial peptide (AMP) family discovered in the skin secretions of Ranidae frogs. The members demonstrate a typical C-terminal ranabox, as well as a diverse range of other structural characteristics. In this study, we identified a novel brevinin-2 peptide from the skin secretion of Sylvirana guentheri, via cloning transcripts, and identifying the expressed mature peptide, in the skin secretion. The confirmed amino acid sequence of the mature peptide was designated brevinin-2GHk (BR2GK). Moreover, as a previous study had demonstrated that the N-terminus of brevinin-2 is responsible for exerting antimicrobial activity, we also designed a series of truncated derivatives of BR2GK. The results show that the truncated derivatives exhibit significantly improved antimicrobial activity and cytotoxicity compared to the parent peptide, except a Pro14 substituted analog. The circular dichroism (CD) analysis of this analog revealed that it did not fold into a helical conformation in the presence of either lipopolysaccharides (LPS) or TFE, indicating that position 14 is involved in the formation of the α-helix. Furthermore, three more analogs with the substitutions of Ala, Lys and Arg at the position 14, respectively, revealed the influence on the membrane disruption potency on bacteria and mammalian cells by the structural changes at this position. Overall, the N-terminal 25-mer truncates demonstrated the potent antimicrobial activity with low cytotoxicity.

A Halotolerant Endo-1,4-β-Xylanase from Aspergillus clavatus with Potential Application for Agroindustrial Residues Saccharification.

The use of non-potable water (such as seawater) is an attractive alternative for water intensive processes such as biomass pretreatment and saccharification steps in the production of biochemicals and biofuels. Identification and application of halotolerant enzymes compatible with high-salt conditions may reduce the energy needed for non-potable water treatment and decrease waste treatment costs. Here we present the biochemical properties of a halotolerant endo-1,4-β-xylanase produced by Aspergillus clavatus in submerged fermentation, using paper sludge (XPS) and sugarcane bagasse (XSCB), and its potential application in the hydrolysis of agroindustrial residues. The peptide mass fingerprint and amino acid sequencing of the XPS and XSCB enzymes showed primary structure similarities with an endo-1,4-β-xylanase from Aspergillus clavatus (XYNA_ASPCL). Both enzyme preparations presented good thermal stability at 50°C and were stable over a wide range of pH and Vmax up to 2450U/mg for XPS. XPS and XSCB were almost fully stable even after 24h of incubation in the presence of up to 3M NaCl, and their activity were not affected by 500mM NaCl. Both enzyme preparations were capable of hydrolyzing paper sludge and sugarcane bagasse to release reducing sugars. These characteristics make this xylanase attractive to be used in the hydrolysis of biomass, particularly with brackish water or seawater.

Characterization and antifungal activity of a plant peptide expressed in the interaction between Capsicum annuum fruits and the anthracnose fungus.

Plant defensins are low molecular weight basic peptides ranging from 5 to 7 kDa, with capacity of inhibiting various pathogens, including fungi. They are present in different tissues of plants, including floral parts and fruits of Capsicum sp. The IIF48 extract, present in immature fruits of Capsicum annuum inoculated with C. gloeosporioides, was able to inhibit up to 100% growth ‘in vitro’ of the fungus Colletotrichum gloeosporioides. The main objective of this work was the purification and antifungal activity characterization of a defense-related plant defensin-like isolated of the IIF48 immature fruits extract. The IIF48 extract was subjected to HPLC purification and 13 fractions were obtained, followed by a tricine gel electrophoresis to obtain the protein profile. The different fractions were submitted to a growth inhibition assay against C. gloeosporioides fungus. Fraction 7 (F7) was the most active causing 73% inhibition. Because of the higher F7 activity and the presence of only a peptide of approximately 5 kDa this fraction was subjected to N-terminal sequencing. F7 fraction was carried out plasma membrane permeabilization assays, induction of intracellular ROS production analysis and investigated mitochondrial membrane potential. The F7 fraction showed significant inhibitory activity on the tested fungus, besides promoting membrane permeabilization, induction of endogenous ROS production in Colletotrichum cells and impairing mitochondrial functionality. The first 18 amino acid sequence of the F7 fraction peptide suggests homology to plant-like defensin and was named IIFF7Ca. We also concluded that IIFF7Ca peptide has an effective antimicrobial action against the fungus C. gloeosporioides.

Identification and Characterization of a New Carboxylesterase 2 Isozyme, mfCES2C, in the Small Intestine of Cynomolgus Monkeys.

In contrast to a single human carboxylesterase 2 (CES2) isozyme (hCE2), three CES2 genes have been identified in cynomolgus monkeys: mfCES2A, mfCES2B, and mfCES2C . Although mfCES2A protein is expressed in several organs, mfCES2B is a pseudogene and the phenotype of the mfCES2C gene has not yet been clarified in tissues. In previous studies, we detected an unidentified esterase in the region of CES2 mobility upon nondenaturing PAGE analysis of monkey intestinal microsomes, which showed immunoreactivity for anti-mfCES2A antibody. The aim of the present study was to identify this unidentified esterase from monkey small intestine. The esterase was separated on nondenaturing PAGE gel and digested in-gel with trypsin. The amino acid sequences of fragmented peptides were analyzed by tandem mass spectrometry. The unidentified esterase was shown to be identical to mfCES2C (XP_015298642.1, predicted from the genome sequence data). mfCES2C consists of 559 amino acid residues and shows approximately 90% homology with mfCES2A (561 amino acid residues). In contrast to the ubiquitous expression of mfCES2A, mfCES2C is only expressed in the small intestine, kidney, and skin. The hydrolytic properties of recombinant mfCES2C, expressed in HEK293 cells, with respect to p-nitrophenyl derivatives, 4-methylumbelliferyl acetate, and irinotecan were similar to those of recombinant mfCES2A. However, mfCES2C showed a hydrolase activity for O-n-valeryl propranolol higher than mfCES2A. It is concluded that the previously unidentified monkey intestinal CES2 is mfCES2C, which shows different hydrolytic properties to mfCES2A, depending on the substrate. SIGNIFICANCE STATEMENT: In the present research, we determined that mfCES2C, a novel monkey CES2 isozyme, is expressed in the small intestine and kidney of the cynomolgus monkey. Interestingly, mfCES2C showed a relatively wide substrate specificity for ester-containing compounds. These findings may, in early stages of drug development, support the use of in vitro-to-in vivo extrapolation for the intestinal hydrolysis of ester drugs in the cynomolgus monkey.

A noncanonical binding site of linezolid revealed via molecular dynamics simulations.

Linezolid, an antibiotic of oxazolidinone family, is a translation inhibitor. The mechanism of its action that consists in preventing the binding of aminoacyl-tRNA to the A-site of the large subunit of a ribosome was embraced on the basis of the X-ray structural analysis of the linezolid complexes with vacant bacterial ribosomes. However, the known structures of the linezolid complexes with bacterial ribosomes poorly explain the linezolid selectivity in suppression of protein biosynthesis, depending on the amino acid sequence of the nascent peptide. In the present study the most probable structure of the linezolid complex with a E.coli ribosome in the A,A/P,P-state that is in line with the results of biochemical studies of linezolid action has been obtained by molecular dynamics simulation methods.

Quantitative Mass Spectrometric Analysis of Autoantibodies as a Paradigm Shift in Autoimmune Serology.

Affecting ~7–9% of individuals worldwide (1), autoimmunity is a relatively common condition that can cause substantial morbidity and mortality (2). However, there are considerable challenges in finding robust and accurate biomarkers for this heterogeneous group of diseases. Serum autoantibodies have served as archetypal diagnostic biomarkers for autoimmune diseases over decades (3). As pathologic species, they can be used to monitor disease activity and treatment responses (4). Most diagnostic laboratory tests for autoantibodies utilize conventional assays such as the solid-phase enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA). All of these assays quantitate amounts of autoantibodies in the bodily fluid but fail to delineate their molecular composition. Multiplex assays have emerged for autoantibody high-throughput screening that enable rapid identification of subsets of patients to facilitate diagnostic and predictive medicine (5). This is particularly important since multiple autoantibodies are often responsible for autoimmune disease (6). However, such conventional assays cannot unravel clonal evolution and dynamic autoimmune responses. Frustratingly, prediction of disease onset and flares with these biomarkers remains suboptimal. Mass spectrometry (MS) is an analytical technique which can identify proteins by determining the amino acid sequence of peptides derived from each protein. MS can also measure changes in relative abundance of specific proteins as a consequence of treatment, and with appropriate standards, quantify absolute abundance. MS has been used previously to analyze specific antibodies or the repertoire of antibodies in order to better understand the dynamics of humoral immune responses in vaccinated animals (7). This technology has been used to characterize autoantibodies in diseases such as systemic lupus erythematosus (SLE) and Sjogren's syndrome (SS) by identifying their immunoglobulin variable region (IgV) subfamily usage and mutational profiles at a molecular level (8). Despite conventional immunoassays determining stability in autoantibody profiles, MS-based quantitative proteomics has been used to uncover the dynamic changes in molecular signatures and levels of autoantibodies as the disease progresses (9). Subtle nuances in the molecular profile of patient autoantibodies can be identified, paving the way for new diagnostic biomarkers that can anticipate the onset or severity of disease before conventional biomarkers or immunoassays (10). This exciting technology hence offers a unique opportunity to identify pathogenic “rogue” and/or protective clonotypes that characterize autoimmune diseases. By deconstructing these clonotypes by quantitative proteomics and establishing a database of clonotypes with their corresponding pathogenicity, this would possibly facilitate identification of at-risk patients for deterioration, or predict response to targeted therapy.

Investigation of the formation mechanism of proline-containing cyclic dipeptide from the linear peptide.

Cyclic dipeptides, 2,5-diketopiperazines (DKPs), are well-known bioactive and taste compounds in food. DKPs have also been reported in various foods and particularly, Pro-containing DKPs (cyclo(-X-Pro)) are more predominant in heated and fermented foods than other type of DKPs. However, the mechanism underlying the preferential formation of Pro-containing DKPs in food remains uncertain. Herein, we attempted to elucidate the effects of reaction conditions and the mechanism of DKPs formation. The reaction conditions (heating time, heating temperature, and pH) and amino acid sequence of the linear peptides were important for the DKPs formation from linear peptides. In addition, Pro-containing DKPs were significantly formed from linear peptides with the second amino acid from the N-terminus being Pro. Based on these results, the underlying mechanism of the enrichment of Pro-containing DKPs in foods was proposed.

Genetic polymorphism of histidine rich protein 2 in Plasmodium falciparum isolates from different infection sources in Yunnan Province, China

BackgroundFailed diagnoses of some falciparum malaria cases by RDTs are constantly reported in recent years. Plasmodium falciparum histidine-rich protein 2 (pfhpr2) gene deficiency has been found to be the major reason of RDTs failure in many countries. This article analysed the deletion of pfhpr2 gene of falciparum malaria cases isolated in Yunnan Province, China.MethodsBlood samples from falciparum malaria cases diagnosed in Yunnan Province were collected. Plasmodium genomic DNA was extracted and the pfhrp2 gene exon2 region was amplified via nested PCR. The haplotype of the DNA sequence, the nucleic acid diversity index (PI) and expected heterozygosity (He) were analyzed. Count PfHRP2 amino acid peptide sequence repeat and its times, and predict the properties of PfHRP2 peptide chain reaction to RDTs testing.ResultsA total of 306 blood samples were collected, 84.9% (259/306) from which pfhrp2 PCR amplification products (gene exon2) were obtained, while the remaining 47 samples were false amplification. The length of the 250 DNA sequences ranged from 345 - 927 bp, with 151 haplotypes, with PI and He values of 0.169 and 0.983, respectively. The length of the PfHRP2 peptide chain translated from 250 DNA sequences ranged from 115 to 309 aa. All peptide chains had more than an amino acid codon deletion. All 250 PfHRP2 strands ended with a type 12 amino acid repeat, 98.0% (245/250) started with a type 1 repetition and 2.0% (5/250) with a type 2 repetition. The detection rate for type 2 duplicates was 100% (250/250). Prediction of RDT sensitivity of PfHRP2 peptide chains based on type 2 and type 7 repeats showed that 9.60% (24/250), 50.0% (125/250), 13.20% (33/250) and 27.20.5% (68/250) of the 250 peptide chains were very sensitive, sensitive, borderline and non-sensitive, respectively.ConclusionThe diversified polymorphism of the pfhrp2 gene deletion from different infection sources in the Yunnan province are extremely complex. The cause of the failure of pfhrp2 exon2 amplification is still to be investigated. The results of this study appeal to Yunnan Province for a timely evaluation of the effectiveness and applicability of RDTs in the diagnosis of malaria.

Bioactive peptides attenuate cardiac hypertrophy and fibrosis in spontaneously hypertensive rat hearts

Alcalase potato protein hydrolysate (APPH), a nutraceutical food, might an have important role in anti-obesity activity. Recent studies from our lab indicated that APPH treatment had lipolysis stimulating activity and identified was an efficient anti-obesity diet ingredient. In this study we aim to investigate the beneficial effects of pure peptide amino acid sequences (DIKTNKPVIF (DI) and IF) from APPH supplement in the regulation of cardiac hypertrophy and fibrosis on spontaneously hypertensive rats (SHR). We examined hematoxylin and eosin staining, Masson's trichrome staining, echocardiographic parameters, serum parameters, hypertrophy, inflammation and fibrotic marker expression to demonstrate efficacy of bioactive peptides in a SHR model. There was a significant upregulation between SHR and bioactive peptides treated groups in left heart weight (LHW), LHW/WHW, LHW/Tibia, LVIDd, and LVd mass. In addition, the bioactive peptides repress the protein expression of hypertrophy markers (BNP, MYH7), inflammation (TLR-4, p-NFkB, TNF-α, IL-6), and fibrotic markers (uPA, MMP-2, TIMP1, CTGF). In summary, these results indicate that DI and IF bioactive peptides from APPH attenuate cardiac hypertrophy, inflammation and fibrosis in the SHR model.

The Isotope-Labeled, MS-Cleavable Cross-Linker Disuccinimidyl Dibutyric Urea for Improved Cross-Linking/Mass Spectrometry Studies.

Previous studies have shown the benefits of the amine-reactive, CID-MS/MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU) for structural proteomics studies via cross-linking/MS (XL-MS). To further facilitate the automation of XL-MS experiments, we synthesized a deuterated (D12) version of the DSBU cross-linker combining the advantages of MS-cleavable linkers and isotope labeling. The rationale of conducting XL-MS with a mixture of unlabeled and stable isotope-labeled DSBU is to obtain characteristic mass differences at the MS level indicating cross-linked species. These cross-linked species can then be selected for fragmentation by collisional activation. At the MS/MS level, the characteristic 26-u doublets arising from cleavage of the central urea group in DSBU confirm the amino acid sequences of cross-linked peptides as well as the exact cross-linking sites. D12-labeled DSBU was tested on three systems with increasing complexity: (i) bovine serum albumin as purified protein, (ii) Escherichia coli ribosome as large, multimeric protein assembly, and (iii) Drosophila embryo extract as complete proteome. We demonstrate the benefits arising from the use of isotope-labeled DSBU for an automated assignment of cross-linked products. Combining isotope labeling and MS cleavability in one cross-linker resulted in higher cross-link identification numbers especially for highly complex protein mixtures.

The Role of an Acidic Peptide in Controlling the Oxidation Process of Walnut Oil

Here, the mechanism of action of an antioxidant peptide rich in acidic amino acid residues in controlling lipid oxidation is discussed. Firstly, in the presence of this peptide, the fluorescence intensity of lipid peroxide in samples of walnut oil was very low, indicating that the peptide prevented the formation of lipid peroxides. Secondly, the production of lipid-derived radicals of oil was reduced by 23% following addition of the anti-oxidative peptide. Thirdly, Raman shifts of the lipid with the anti-oxidative peptide showed that acidic amino acid residues of the peptide were involved in delaying lipid oxidation. Finally, seven peptide inhibitors were synthesized with variations to the amino acid sequence of the original peptide, and Glu–Asp was proven to enhance the peptide’s superoxide anion radical scavenging activity and decrease the formation of linoleic acid peroxides. Our findings emphasize the potential value of acidic amino acid residues in protecting unsaturated fatty acids from oxidation.

Angiotensin I‐converting enzyme inhibitory peptides produced from tuna cooking juice hydrolysate by continuous enzymatic membrane reactor

Angiotensin I-converting enzyme (ACE) inhibitory peptides were derived from tuna cooking juice (TCJ) hydrolysis by alcalase in the continuous enzymatic membrane reactor (cEMR) coupling with 1kDa MWCO membrane. The permeated sample from cEMR for 510min of hydrolysis was purified by size exclusion chromatography in Sephadex G-25 column. A fraction exhibited the highest ACE inhibitory activity was further separated by RP-HPLC, resulting two fractions showed highest ACE inhibitory activities. The molecular weight (MW) and amino acid sequences of peptides from both fractions were determined using LC-MS/MS. Two potential ACE inhibitory peptides were obtained and showed molecular weight of 959.46 and 1,141.29Da. PRACTICAL APPLICATIONS: Tuna cooking juice (TCJ) usually was either used as protein source of feed meal or directly discharged to wastewater treatment system. However, it contains water-soluble proteins in a group of sarcoplasmic protein, which is small water-soluble proteins and easily hydrolyzed to small peptides. In this study, the active peptides, angiotensin I-converting enzyme inhibitory peptides (MW of 959.46 and 1,141.29Da), obtained from TCJ hydrolysate and identified by LC-MS/MS would be a beneficial ingredient for nutraceuticals and functional food against hypertension.

Analysis of glycoside hydrolases from oat (Avena sativa) seedling extract.

The abundance and the diversity of oligo- and polysaccharides provide a wide range of biological roles attributed either to these carbohydrates or to their relevant enzymes, i.e., the glycoside hydrolases (GHs). The biocatalysis by these families of enzymes is highly attractive for the generation of products used in potential applications, e.g., pharmaceuticals and food industries. It is thus very important to extract and characterize such enzymes, particularly from plant tissues. In this study, we characterized novel sequences of class I chitinases from seedlings extract of the common oat (Avena sativa L.) using proteomics and sequence-structure-function analysis. These enzymes, which belong to the GH19 family of protein, were extracted from oat and identified using SDS-PAGE, trypsin digestion, LC-MS-MS, and sequence-structure-function analysis. The amino acid sequences of the oat tryptic peptides were used to identify cDNAs from the Avena sativa databases of the expressed sequence tags (ESTs) and transcriptome shotgun assembly (TSA). Based upon the Avena sativa sequences of ESTs and TSA, at least 4 predicted genes that encoded oat class I chitinases were identified and reported. The structural characterization of the oat sequences of chitinases provided valuable insights to the context.

Interpretation of Tandem Mass Spectra of Posttranslationally Modified Peptides.

Tandem mass spectrometry provides a sensitive means of analyzing the amino acid sequence of peptides and modified peptides by providing accurate mass measurements of precursor and fragment ions. Modern mass spectrometry instrumentation is capable of rapidly generating many thousands of tandem mass spectra and protein database search engines have been developed to match the experimental data to peptide candidates. In most studies there is a schism between discarding perfectly valid data and including nonsensical peptide identifications-this is currently managed by establishing a false discovery rate (FDR) but for modified peptides it calls for an understanding of tandem mass spectrometry data. Manual evaluation of the data and perhaps experimental cross-checking of the MS data can save many months of experimental work trying to do biological follow-ups based on erroneous identifications. Especially for posttranslationally modified peptides there is a need for careful consideration of the data because search algorithms seldom have been optimized for the identification of modified peptides and because there are many pitfalls for the unwary. This chapter describes some of the issues that should be considered when interpreting and validating tandem mass spectra and gives some useful tables to aid in this process.

DeepMSPeptide: peptide detectability prediction using deep learning.

The protein detection and quantification using high-throughput proteomic technologies is still challenging due to the stochastic nature of the peptide selection in the mass spectrometer, the difficulties in the statistical analysis of the results and the presence of degenerated peptides. However, considering in the analysis only those peptides that could be detected by mass spectrometry, also called proteotypic peptides, increases the accuracy of the results. Several approaches have been applied to predict peptide detectability based on the physicochemical properties of the peptides. In this manuscript, we present DeepMSPeptide, a bioinformatic tool that uses a deep learning method to predict proteotypic peptides exclusively based on the peptide amino acid sequences. DeepMSPeptide is available at https://github.com/vsegurar/DeepMSPeptide. Supplementary data are available at Bioinformatics online.

XMAn v2-a database of Homo sapiens mutated peptides.

The 'Unknown Mutation Analysis (XMAn)' database is a compilation of Homo sapiens mutated peptides in FASTA format, that was constructed for facilitating the identification of protein sequence alterations by tandem mass spectrometry detection. The database comprises 2 539 031 non-redundant mutated entries from 17 599 proteins, of which 2 377 103 are missense and 161 928 are nonsense mutations. It can be used in conjunction with search engines that seek the identification of peptide amino acid sequences by matching experimental tandem mass spectrometry data to theoretical sequences from a database. XMAn v2 can be accessed from github.com/lazarlab/XMAnv2. Supplementary data are available at Bioinformatics online.

Abstract P1103: Angiotensin II Type I Receptor Agonistic Autoantibody Blockade Improves Hypertension and Immune Activation in Postpartum Preeclamptic Rats

Women with preeclampsia (PE) have a risk of developing cardiovascular diseases (CVD) later in life. The angiotensin II type I receptor agonistic autoantibodies (AT1-AAs) are elevated in women with PE, PE women 2 years post-partum (PP), and the reduced uterine perfusion pressure (RUPP) rat model of PE. Blockade of the AT1-AA by using a specific binding seven amino acid peptide sequence (7AA) improves the pathology of PE in RUPP rats. The long-term effects of AT1-AA inhibition on blood pressure, NK cell activation, heart mitochondria (mt) proteins, and CVD in the RUPP model PP is unknown. Therefore, we hypothesized that PP RUPP rats have elevated blood pressure, NK cell activation, and changes in heart mt proteins, which will be prevented in RUPP rats administered the 7AA during pregnancy. Methods: Pregnant Sprague Dawley rats were divided into groups; normal pregnant (NP) (n=7), RUPP (n=10), and RUPP+7AA (n=9). Gestational day 14, RUPP surgery was performed and 7AA (2 μg/ml) administered via minipump. Results: At 8 and 10 weeks (wks) PP, blood pressure (MAP), blood, and hearts were collected. NK cells were quantified by flow cytometry. At 8 wks PP, MAP was elevated in RUPP vs. NP (130±2 vs. 123±4 mmHg, ns), and RUPP+7AA (124±4 mmHg) treatment prevented this increase. At 10 wks, MAP was elevated in RUPP vs NP (133±5 vs. 120±5 mmHg, p=0.08), with a significant decrease in MAP in RUPP+7AA (107±6 mmHg) vs. RUPP (p<0.05). Total circulating NK cells were increased in RUPP vs NP (45±9. vs. 29± 8% gated cells, ns), which was prevented in RUPP+7AA (19±16 % gated cells) at 8 wks PP. Hearts were enlarged with RUPP vs NP (0.41±0.04 vs. 0.36±0.02g/100gBW, ns), which was normalized in RUPP+7AA (0.34±0.02g/100gBW). Previous studies show that during pregnancy complex IV is significantly lower along with a decrease mt function in RUPP vs NP. Complex IV mt proteins in the heart were elevated in RUPP+7AA vs. RUPP (5.5±1.7 vs. 3.0±0.2 AU, ns). Conclusion: In summary, PP PE rats have an increase in MAP, NK cells, and larger hearts. AT1-AA inhibition restores complex IV mt levels and improves HTN, immune activation, and cardiac hypertrophy PP. This study highlights the importance of AT1-AA inhibition during PE to prevent CVD later in life. Supported: AHA18CDA34110264

Evaluating the Bioactivity of a Novel Antimicrobial and Anticancer Peptide, Dermaseptin-PS4(Der-PS4), from the Skin Secretion of Phyllomedusa sauvagii.

Dermaseptins belonging to a large family of cationic membrane-disruption antimicrobial peptides display extensive antibacterial and antiproliferative activities depending on a coil-to-helix transition and the specific structural parameters. Herein, a novel dermaseptin peptide named Der-PS4 was discovered from the skin secretion of the waxy monkey tree frog, Phyllomedusa sauvagii. The complementary DNA (cDNA)-encoding precursor was obtained relying on “shotgun” cloning, and afterwards, a mature peptide amino acid sequence was identified by reverse-phase high performance liquid chromatography (RP-HPLC) and MS/MS. Specimens were chemically synthesized and applied for further functional studies. Structural analysis demonstrated a higher α-helical content in the membrane-mimetic environment compared with that in the ammonium acetate/water circumstance. Der-PS4 displayed a broad spectrum of antimicrobial activities against tested pathogenic microorganisms, however, exhibiting slight membrane-damaging effectiveness towards horse red blood cells. Coincident with the inhibitory activities on pathogens, Der-PS4 also showed considerable biofilm eradicating impact. Also, Der-PS4 penetrated cell membrane in a relative short period under each minimum bactericidal concentration. In addition, Der-PS4 possessed antiproliferative capacity against five cancer cell lines, while presenting slight suppressing effect on human microvascular endothelial, HMEC-1. These findings provide a promising insight for the discovery and development of novel drugs from a natural source.

Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)*[S

Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody's epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human β-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown.

Structural and functional characterisation of a novel peptide from the Australian sea anemone Actinia tenebrosa

Sea anemone venoms have long been recognised as a rich source of peptides with interesting pharmacological and structural properties. Our recent transcriptomic studies of the Australian sea anemone Actinia tenebrosa have identified a novel 13-residue peptide, U-AITx-Ate1. U-AITx-Ate1 contains a single disulfide bridge and bears no significant homology to previously reported amino acid sequences of peptides from sea anemones or other species. We have produced U-AITx-Ate1 using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The solution structure of U-AITx-Ate1 was determined based on two-dimensional nuclear magnetic resonance spectroscopic data. Diffusion-ordered NMR spectroscopy revealed that U-AITx-Ate1 was monomeric in solution. Perturbations in the 1D 1H NMR spectrum of U-AITx-Ate1 in the presence of dodecylphosphocholine micelles together with molecular dynamics simulations indicated an interaction of U-AITx-Ate1 with lipid membranes, although no binding was detected to 100% POPC and 80% POPC: 20% POPG lipid nanodiscs by isothermal titration calorimetry. Functional assays were performed to explore the biological activity profile of U-AITx-Ate1. U-AITx-Ate1 showed no activity in voltage-clamp electrophysiology assays and no change in behaviour and mortality rates in crustacea. Moderate cytotoxic activity was observed against two breast cancer cell lines.