RSSRoles of tRNA in cell wall biosynthesis.
Source
Department of Microbiology, Ohio State University, Columbus, OH, USA.
Abstract
Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorterpeptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes they impart on the bacterium differ greatly. WIREs RNA 2012. doi: 10.1002/wrna.1108 For further resources related to this article, please visit the WIREs website.
Copyright © 2012 John Wiley & Sons, Ltd.
- PMID:
- 22262511
- [PubMed - as supplied by publisher]
Metallopolymer-Peptide Hybrid Materials: Synthesis and Self-Assembly of Functional, Polyferrocenylsilane-Tetrapeptide Conjugates.
Source
School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS (UK), Fax: (+44) 117-925-1295, Fax: (+44) 117-929-0509.
Abstract
Conjugates of poly(ferrocenyldimethylsilane) (PFDMS) with Ac-(GA)(2) -OH, Ac-A(4) -OH, Ac-G(4) -OH and Ac-V(4) -OH have been prepared by reaction of the tetrapeptide units with the amino-terminated metallopolymer. The number average degree of polymerisation (DP(n) ) of the PFDMS was approximately 20 and comparable materials with shorter (DP(n) ≈10) and/or amorphous chains have been prepared by the same procedure. Poly(ferrocenylethylmethylsilane) (PFEMS) was employed for the latter purpose. All conjugates were characterised by GPC, MALDI-TOF MS, NMR and IR spectroscopy. With the exception of Ac-V(4) -PFDMS(20) , all materials exhibited some anti-parallel β-sheet structure in the solid state. The self-assembly of the conjugates was studied in toluene by DLS. The vast majority of the materials, irrespective of peptide sequence or chain crystallinity, afforded fibres consisting of a peptidic core surrounded by a PFS corona. These fibres were found in the form of cross-linked networks by TEM and AFM. The accessibility of the chemically reducing PFS corona has been demonstrated by the localised formation of silver nanoparticles on the surface of the fibres.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- PMID:
- 22262498
- [PubMed - as supplied by publisher]
In vitro and in vivo evaluation of a (64)Cu-labeled NOTA-Bn-SCN-Aoc-bombesin analogue in gastrin-releasing peptide receptor expressing prostate cancer.
Source
Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108, USA.
Abstract
INTRODUCTION:
Bombesin (BN) is an amphibian peptide that binds to the gastrin-releasing peptide receptor (GRPR). It has been demonstrated that BN analogues can be radiolabeled for potential diagnosis and treatment of GRPR-expressing malignancies. Previous studies have conjugated various chelators to the eight C-terminal amino acids of BN [BN(7-14)] for radiolabeling with (64)Cu. Recently, (1,4,7-triazacyclononane-1,4,7-triacetic acid) (NOTA) has been evaluated as the five-coordinate (64)Cu complex, with results indicating GRPR-specific tumor uptake. This study aimed to conjugate S-2-(4-isothiocyanatobenzyl)-NOTA (p-SCN-Bn-NOTA) to BN(7-14) such that it could form a six-coordinate complex with (64)Cu and to evaluate the resulting peptide.
METHODS:
p-SCN-NOTA was conjugated to 8-aminooctanoic acid (Aoc)-BN(7-14) in solution to yield NOTA-Bn-SCN-Aoc-BN(7-14). The unlabeled peptide was evaluated in a cell binding assay using PC-3 prostate cancer cells and (125)I-Tyr(4)-BN to determine the IC(50) value. The peptide was radiolabeled with (64)Cu and evaluated for internalization into PC-3 cells and for tumor uptake in mice bearing PC-3 xenografts using biodistribution and micro-positron emission tomography imaging studies.
RESULTS:
The binding assay demonstrated that NOTA-Bn-SCN-Aoc-BN(7-14) bound with high affinity to GRPR with an IC(50) of 1.4 nM. The radiolabeled peptide demonstrated time-dependent internalization into PC-3 cells. In vivo, thepeptide demonstrated tumor-specific uptake and imaging that were comparable to those of previously reported (64)Cu-labeled BN analogues.
CONCLUSIONS:
These studies demonstrate that (64)Cu-NOTA-Bn-SCN-Aoc-BN(7-14) binds to GRPR-expressing cells and that it can be used for imaging of GRPR-expressing prostate cancer.
Copyright © 2011 Elsevier Inc. All rights reserved.
- PMID:
- 22261146
- [PubMed - as supplied by publisher]
Bombesin analogues for gastrin-releasing peptide receptor imaging.
Source
Department of Radiology, University of Missouri School of Medicine, Columbia, Missouri 65211, USA.
Abstract
OBJECTIVES:
The present study describes the design and development of a series of new bombesin (BBN) antagonistpeptide ligands of the form [(64)Cu-(NO2A-X-D-Phe(6)-BBN(6-13)NHEt)], where Cu-64=a positron emitting radiometal; NO2A=1,4,7-triazacyclononane-1,4-diacetic acid; X=6-amino hexanoic acid, 8-amino octanoic acid or 9-Aminononanoic acid; and BBN(6-13)NHEt=Gln-Trp-Ala-Val-Gly-His-Leu-NHEt, an antagonist analogue of bombesin peptide for specific targeting of the gastrin-releasing peptide receptor (GRPR).
METHODS:
[NO2A-X-D-Phe(6)-BBN(6-13)NHEt] conjugates were manually conjugated with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the resulting conjugates were labeled with (64)Cu to yield [(64)Cu-(NO2A-X-D-Phe(6)-BBN(6-13)NHEt)]. The metallated and nonmetallated conjugates were purified via reversed-phase high-performance liquid chromatography and characterized by electrospray ionization-mass spectrometry.
RESULTS:
Competitive displacement binding assays displayed nanomolar binding affinities toward human GRPR for all of the newly formed peptide analogues. Biodistribution studies showed very high uptake and retention of tumor-associated radioactivity in PC-3 (a prostate tumor model known to express the GRPR) tumor-bearing rodent models. The radiolabeled conjugates also exhibited rapid urinary excretion and very high tumor to background ratios. Micro-positron emission tomography (PET) molecular imaging investigations showed clear visualization of tumors in female PC-3 tumor-bearing mice 15 h postinjection.
CONCLUSION:
The biodistribution and molecular imaging study suggests that these conjugates can be considered as potential PET tracer candidates for the diagnosis of GRPR-positive tumors in human patients.
Published by Elsevier Inc.
- PMID:
- 22261143
- [PubMed - as supplied by publisher]
Mapping of B cell epitopes on desmoglein 3 in pemphigus vulgaris patients by the use of overlapping peptides.
Source
Department of Dermatology, University of Lübeck, Lübeck, Germany.
Abstract
BACKGROUND:
Pemphigus vulgaris (PV) is a severe autoimmune blistering disease associated with autoantibodies to desmoglein 3 (Dsg 3), a transmembrane glycoprotein of the cadherin family. Previous studies mainly focused on the mapping of conformational epitopes of Dsg 3 using recombinant fragments of Dsg 3 and competition ELISA.
OBJECTIVE:
Here, we performed a mapping of linear B cell epitopes on Dsg 3 in PV patients by the use of overlapping synthetic peptides.
METHODS:
A set of 254 overlapping synthetic peptides of 14 amino acids length covering the entire Dsg 3 extracellular domain was generated. Sera of patients with active PV (n=10) and healthy volunteers (n=10) were tested for IgG reactivity with the 254 peptides by ELISA. Testing each peptide separately, 7 major antigenic sites were identified. In order to validate these reactivities, 7 corresponding peptides of 13-33 amino acids in length were generated and employed by ELISA. Additional sera of active PV patients (n=17) and healthy volunteers (n=20) were tested and the most reactive peptide was used to specifically purify anti-Dsg 3 antibodies from PV sera (n=3).
RESULTS:
The major autoantibody reactivity in PV sera was mapped to amino acids 333-356 within the EC3 domain. Purifying patients IgG using the identified peptide, however, failed to induce acantholysis in keratinocyte dissociation assay.
CONCLUSION:
We conclude that linear epitopes do not play a major pathogenic role in human PV.
Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
- PMID:
- 22261006
- [PubMed - as supplied by publisher]
Amino acid mixture acutely improves the glucose tolerance of healthy overweight adults.
Source
Exercise Physiology and Metabolism Laboratory, Department of Kinesiology and Health Education, University of Texas at Austin, Austin, TX 78712-0360, USA.
Abstract
Certain amino acids have been reported to influence carbohydrate metabolism and blood glucose clearance, as well as improve the glucose tolerance in animal models. We hypothesized that an amino acid mixture consisting of isoleucine and 4 additional amino acids would improve the glucose response of healthy overweight men and women to an oral glucose tolerance test (OGTT). Twenty-two overweight healthy subjects completed 2 OGTTs after consuming 2 different test beverages. The amino acid mixture beverage (CHO/AA) consisted of 0.088 g cystine 2HCl, 0.043 g methionine, 0.086 g valine, 12.094 g isoleucine, 0.084 g leucine, and 100 g dextrose. The control beverage (CHO) consisted of 100 g dextrose only. Venous blood samples were drawn 10 minutes before the start of ingesting the drinks and 15, 30, 60, 120, and 180 minutes after the completion of the drinks. During the OGTT, the plasma glucose response for the CHO/AA treatment was significantly lower than that of the CHO treatment (P < .01), as was the plasma glucose area under the curve (CHO/AA 806 ± 31 mmol/L·3 hours vs CHO 942 ± 40 mmol/L·3 hours). Differences in plasma glucose between treatments occurred at 30, 60, 120, and 180 minutes after supplement ingestion. Plasma glucagon during the CHO/AA treatment was significantly higher than during the CHO treatment. However, there were no significant differences in plasma insulin or C-peptide responses between treatments. These results suggest that the amino acid mixture lowers the glucose response to an OGTT in healthy overweight subjects in an insulin-independent manner.
Copyright © 2012 Elsevier Inc. All rights reserved.
- PMID:
- 22260861
- [PubMed - in process]
Comprehensive Conformational Studies of Five Tripeptides and a Deduced Method for Efficient Determinations of Peptide Structures.
Abstract
Thorough searches on the potential energy surfaces of five tripeptides, GGG, GYG, GWG, TGG and MGG, were performed by considering all possible combinations of the bond rotational degrees of freedom with a semi-empirical and ab initio combined computational approach. Structural characteristics of the obtained stable tripeptide conformers were carefully analyzed. Conformers of the five tripeptides were found to be closely connected with conformers of their constituting dipeptides and amino acids. A method for finding all important tripeptide conformers by optimizing a small number of trial structures generated by suitable superposition of the parent amino acid and dipeptide conformers is thus proposed. Applying the method to another five tripeptides, YGG, FGG, WGG, GFA and GGF, studied before shows that the new approach is both efficient and reliable by providing the most complete ensembles of tripeptide conformers. The method is further generalized for application to larger peptides by introducing the breeding and mutation concepts in a genetic algorithm way. The generalized method is verified to be capable of finding tetrapeptide conformers with secondary structures of strands, helices and turns which are highly populated in larger peptides. This show some promise for the proposed method to be applied for the structural determination of larger peptides.
- PMID:
- 22260814
- [PubMed - as supplied by publisher]
Charge Transfer in Model Peptides - Obtaining Marcus Parameters from Molecular Simulation.
Abstract
Charge transfer within and between biomolecules remains a highly active field of biophysics. Due to the complexities of real systems, model compounds are a useful alternative to study the mechanistic fundamentals of charge transfer. In recent years, such model experiments have been underpinned by molecular simulation methods as well. In this work, we study electron hole transfer in helical model peptides by means of molecular dynamics simulations. A theoretical framework to extract Marcus parameters of charge transfer from simulations is presented. We find that the peptidesform stable helical structures with sequence dependent small deviations from ideal PPII helices. We identify direct exposure of charged side chains to solvent as a cause of high reorganisation energies, significantly larger than typical for electron transfer in proteins. This, together with small direct couplings, makes long-range superexchange electron transport in this system very slow. In good agreement to experiment, direct transfer between the terminal amino acid side chains can be dicounted in favor of a two-step hopping process if appropriate bridging groups exist.
- PMID:
- 22260641
- [PubMed - as supplied by publisher]
Binding of a truncated form of Lecithin : retinol acyltransferase and its N- and C-terminal peptides to lipid monolayers.
Abstract
Lecithin retinol acyltransferase (LRAT) is a 230 amino acids membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. A truncated form of LRAT (tLRAT), which contains the residues required for catalysis but which is lacking the N- and C-terminal hydrophobic segments, was produced to study its membrane binding properties. Measurements of the maximum insertion pressure of tLRAT, which is higher than the estimated lateral pressure of membranes, and the positive synergy factor a argue in favor of a strong binding of tLRAT to phospholipid monolayers. Moreover, the binding, secondary structure and orientation of the peptides corresponding to the N- and C-terminal hydrophobic segments of LRAT have been studied by circular dichroism and polarization-modulation infrared reflection absorption spectroscopy in monolayers. The results show that these peptidesspontaneously bind to lipid monolayers and adopt an alpha helical secondary structure. On the basis of these data, a new membrane topology model of LRAT is proposed where its N- and C-terminal segments allow to anchor this protein to the lipid bilayer.
- PMID:
- 22260449
- [PubMed - as supplied by publisher]
Comparative analysis of recombinant Human Papillomavirus 8 L1 production in plants by a variety of expression systems and purification methods.
Source
Istituto di Virologia Vegetale, CNR, Strada delle Cacce, Torino, Italy.
Abstract
Human papillomavirus 8 (HPV-8), one of the high-risk cutaneous papillomaviruses (cHPVs), is associated with epidermodysplasia verruciformis and nonmelanoma skin cancer in immuno-compromised individuals. Currently, no vaccines against cHPVs have been reported; however, recent studies on cross-neutralizing properties of their capsid proteins (CP) have fostered an interest in vaccine production against these viruses. We examined the potential of producing HPV-8 major CP L1 in Nicotiana benthamiana by agroinfiltration of different transient expression vectors: (i) the binary vector pBIN19 with or without silencing suppressor constructs, (ii) the nonreplicating Cowpea mosaic virus-derived expression vector pEAQ-HT and (iii) a replicating Tobacco mosaic virus (TMV)-based vector alone or with signalpeptides. Although HPV-8 L1 was successfully expressed using pEAQ-HT and TMV, a 15-fold increase was obtained with pEAQ-HT. In contrast, no L1 protein could be immune detected using pBIN19 irrespective of whether silencing suppressors were coexpressed, although such constructs were required for identifying L1-specific transcripts. A fourfold yield increase in L1 expression was obtained when 22 C-terminal amino acids were deleted (L1ΔC22), possibly eliminating a nuclear localization signal. Electron microscopy showed that plant-made HPV-8 L1 proteins assembled in appropriate virus-like particles (VLPs) of T = 1 or T = 7 symmetry. Ultrathin sections of L1ΔC22-expressing cells revealed their accumulation in the cytoplasm in the form of VLPs or paracrystalline arrays. These results show for the first time the production and localization of HPV-8 L1 protein in planta and its assembly into VLPs representing promising candidate for potential vaccine production.
© 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.
- PMID:
- 22260326
- [PubMed - as supplied by publisher]
Cyclo-linopeptide B methanol tris-olvate.
Abstract
The title compound, C(56)H(83)N(9)O(9)S·3CH(3)OH, is a methanol tris-olvate of the cyclo-linopeptide cyclo(Met(1)-Leu(2)-Ile(3)-Pro(4)-Pro(5)-Phe(6)-Phe(7)-Val(8)-Ile(9)) (henceforth referred to as CLP-B), which was isolated from flaxseed oil. All the amino acid residues are in an l-configuration based on the CORN rule. The cyclic nona-peptideexhibits eight trans peptide bonds and one cis peptide bond observed between the two proline residues. The conformation is stabilized by an α-turn and two consecutive β-turns each containing a N-H⋯O hydrogen bond between the carbonyl group O atom of the first residue and the amide group H atom of the fourth (α-turn) or the third residue (β-turns), repectively. In the crystal, the components of the structure are linked by N-H⋯O and O-H⋯O hydrogen bonds into chains parallel to the a axis.
- PMID:
- 22259553
- [PubMed - in process]
O-glycoprotein biosynthesis: site localization by edman degradation and site prediction based on random Peptide substrates.
Source
Department of Pediatrics and Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, OH, USA, txg2@cwru.edu.
Abstract
The characterization of mucin-type O-glycosylation is fraught with extreme difficulty at almost every level of analysis: from difficulties in obtaining glycopeptides suitable for study, their structural heterogeneity, lack of broad acting glycosidase tools capable of simplifying the glycans, and finally the vast complexity of performing analysis on multiply glycosylated glycopeptides. This, along with a lack of known peptide sequence motif(s) for the transferases that initiate mucin-type O-glycosylation, significantly hinders our understanding of mucin-type O-glycosylation at almost every level from their biosynthesis to their biological and biophysical properties. In this chapter, the use of partial chemical deglycosylation coupled with Edman amino acid sequencing is described to quantify sites of O-glycosylation. In addition, the use of oriented random peptide substrates is described for providing the specificities of the polypeptide α-N-acetylgalactosaminyltransferases, which can be used to estimate transferase-specific sites of O-glycosylation.
Contributions of Individual Amino Acid Residues to the Endogenous CLV3 Function in Shoot Apical Meristem Maintenance in Arabidopsis.
Source
Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Fragrant Hill, Beijing 100093, China.
Abstract
As a peptide hormone, CLV3 restricts the stem cell number in shoot apical meristem (SAM) by interacting with CLV1/CLV2/CRN/RPK2 receptor complexes. To elucidate how the function of the CLV3 peptide in SAM maintenance is established at the amino acid (AA) level, alanine substitutions were performed by introducing point mutations to individual residues in the peptide-coding region of CLV3 and its flanking sequences. Constructs carrying such substitutions, expressed under the control of CLV3 regulatory elements, were transformed to the clv3-2 null mutant to evaluate their efficiencies in complementing its defects in SAMs in vivo. These studies showed that aspartate-8, histidine-11, glycine-6, proline-4, arginine-1, and proline-9, arranged in an order of importance, were critical, while threonine-2, valine-3, serine-5, and the previously assigned hydroxylation and arabinosylation residue proline-7 were trivial for the endogenous CLV3 function in SAM maintenance. In contrast, substitutions of flanking residues did not impose much damage on CLV3. Complementation of different alanine-substituted constructs was confirmed by measurements of the sizes of SAMs and the WUS expression levels in transgenic plants. These studies established a complete contribution map of individual residues in the peptide-coding region of CLV3 for its function in SAM, which may help to understand peptide hormones in general.
A novel family of peptides with potent activity against influenza A viruses.
Source
University of Edinburgh.
Abstract
The emergence of drug resistant strains of influenza virus has catalyzed a search for new antiviral agents to supplement or replace existing drugs. Following the success of the HIV entry blocker Enfuvirtide, there has been a resurgence of interest in peptide based antivirals. In this paper we report on the discovery of a novel family of peptides (FluPep, FP) that function as inhibitors of influenza A virus infection. The prototype peptide (FP1, also known as Tkip) interacts with haemagglutinin and inhibits the binding of the virus to cell membranes. Using a plaque reduction assay we have demonstrated that a variety of influenza A virus subtypes (including H1N1, H3N2 H5N1) are inhibited by FluPep and its derivatives at nanomolar concentrations. By truncating FluPep we have identified a minimal sequence of 6 amino acids that binds to HA and inhibits infection. Using a mouse model of intranasal influenza virus infection we observed potent inhibition of virus infection when peptide is given at the time of virus administration. These data indicate that FluPep is a highly effective anti-influenza agent with the potential to translate to the clinic.
Retargeting of rat parvovirus H-1PV to cancer cells through genetic engineering of the viral capsid.
Source
Tumour Virology Division F010.
Abstract
The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side-effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose, away from the tumour target. Hence, targeting H-1PV entry specifically to tumour cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. Then, we genetically engineered the H-1PV capsid in order to improve its affinity for human tumour cells. By analogy with the resolved crystal structure of the closely related parvovirus MVM, we developed an in silico 3D model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the twofold axis of symmetry of the capsid, within the so-called "dimple" region. In situ mutagenesis of these residues significantly reduced binding and entry of H-1PV into permissive cells. We then engineered the entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its threefold axis spike. This peptide binds α(v)β(3) and α(v)β(5) integrins, which are over-expressed in cancer cells and growing blood vessels. Insertion of thepeptide rescued viral infectivity towards cells over-expressing α(v)β(5) integrins resulting in efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.
The Serine-Proline Turn: A Novel Hydrogen-Bonded Template for Designing Peptidomimetics.
Source
Center for Advanced Drug Research (CADRE), SRI International, 140 Research Drive, Harrisonburg, Virginia 22802, United States.
Abstract
Serine-Proline (SP) dipeptide motifs have been shown to form unique hydrogen-bonding patterns in protein crystal structures. Peptides were designed to mimic these patterns by forming the 6 + 10 and the 9 + 10 hydrogen-bonded rings. Factors that contribute to the formation of SP turns include controlling backbone flexibility and amino acid chirality along with creating a hydrophobic environment around the intramolecular hydrogen bonds.
Chemical deamidation: a common pitfall in large-scale N-linked glycoproteomic mass spectrometry-based analyses.
Abstract
N-linked glycoproteins are involved in several diseases and are important as potential diagnostic molecules for biomarker discovery. Therefore, it is important to provide sensitive and reliable analytical methods to identify not only the glycoproteins but also the sites of glycosylation. Recently, numerous strategies to identify N-linked glycosylation sites have been described. These strategies have been applied to cell lines and several tissues with the aim of identifying many hundreds (or thousands) of glycosylation events. With high-throughput strategies however, there is always the potential for false positives. The confusion arises since the protein N-glycosidase F (PNGase F) reaction used to separate N-glycans from formerly glycosylated peptides catalyses the cleavage and deamidates the asparagine residue. This is typically viewed as beneficial since it acts to highlight the modification site. We have evaluated this common large-scale N-linked glycoproteomic strategy and proved potential pitfalls using Escherichia coli as a model organism, since it lacks the N-glycosylation machinery found in mammalian systems and some pathogenic microbes. After isolation and proteolytic digestion of E. coli membrane proteins, we investigated the presence of deamidated asparagines. The results show the presence of deamidated asparagines especially with close proximity to a glycine residue or other small amino acid, as previously described for spontaneous in vivo deamidation. Moreover, we have indentified deamidated peptides with incorporation of 18O, showing the pitfalls of glycosylation site assignment based on deamidation of asparagine induced by PNGase F in 18O-water in large-scale analyses. These data experimentally prove the need for more caution in assigning glycosylation sites and "new" N-linked consensus sites based on common N-linked glycoproteomics strategies without proper control experiments. Beside showing the spontaneous deamidation we provide alternative methods for validation that should be used in such experiments.
Dietary proteins as determinants of metabolic and physiologic functions of the gastrointestinal tract.
Source
Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, M5S 3E2, Canada; Email: alireza.jahanmihan@utoronto.ca (A.J.-M.); bohdan.luhovyy@utoronto.ca (B.L.L.); dalia.elkhoury@utoronto.ca (D.E.K.).
Abstract
Dietary proteins elicit a wide range of nutritional and biological functions. Beyond their nutritional role as the source ofamino acids for protein synthesis, they are instrumental in the regulation of food intake, glucose and lipid metabolism, blood pressure, bone metabolism and immune function. The interaction of dietary proteins and their products of digestion with the regulatory functions of the gastrointestinal (GI) tract plays a dominant role in determining the physiological properties of proteins. The site of interaction is widespread, from the oral cavity to the colon. The characteristics of proteins that influence their interaction with the GI tract in a source-dependent manner include their physico-chemical properties, their amino acid composition and sequence, their bioactive peptides, their digestion kinetics and also the non-protein bioactive components conjugated with them. Within the GI tract, these products affect several regulatory functions by interacting with receptors releasing hormones, affecting stomach emptying and GI transport and absorption, transmitting neural signals to the brain, and modifying the microflora. This review discusses the interaction of dietary proteins during digestion and absorption with the physiological and metabolic functions of the GI tract, and illustrates the importance of this interaction in the regulation of amino acid, glucose, lipid metabolism, and food intake.
Immunological Evaluation of Lipopeptide Group A Streptococcus (GAS) Vaccine: Structure-Activity Relationship.
Source
The University of Queensland, School of Chemistry and Molecular Biosciences (SCMB), St. Lucia, Queensland, Australia.
Abstract
Streptococcus pyogenes (group A streptococcus, GAS) is a Gram-positive bacterial pathogen responsible for a wide variety of diseases. To date, GAS vaccine development has focused primarily on the M-protein. The M-protein is highly variable at the amino (N)-terminus (determining serotype) but is conserved at the carboxyl (C)-terminus. Previously a 29amino acid peptide (named J14) from the conserved region of the M-protein was identified as a potential vaccine candidate. J14 was capable of eliciting protective antibodies that recognized many GAS serotypes when co-administered with immuno-stimulants. This minimal epitope however showed no immunogenicity when administered alone. In an attempt overcome this immunological non-responsiveness, we developed a self-adjuvanting vaccine candidate composed of three components: the B-cell epitope (J14), a universal helper T-cell epitope (P25) and a lipid moiety consisting of lipoamino acids (Laas) which target Toll-like receptor 2 (TLR2). Immunological evaluation in B10.BR (H-2k) mice demonstrated that the epitope attachment to the point of lipid moiety, and the length of the Laa alkyl chain have a profound effect on vaccine immunogenicity after intranasal administration. It was demonstrated that a vaccine featuring C-terminal lipid moiety containing alkyl chains of 16 carbons, with P25 located at the N-terminus, and J14 attached to the side chain of a central lysine residue was capable of inducing optimal antibody response. These findings have considerable relevance to the development of a broad spectrum J14-based GAS vaccine and in particular provided a rational basis for peptide vaccine design based on this self-adjuvanting lipopeptide technology.
Novel protocol for the chemical synthesis of crustacean hyperglycemic hormone analogues - an efficient experimental tool for studying their functions.
Source
Department of Life Sciences, University of Trieste, Trieste, Italy.
Abstract
The crustacean Hyperglycemic Hormone (cHH) is present in many decapods in different isoforms, whose specific biological functions are still poorly understood. Here we report on the first chemical synthesis of three distinct isoforms of the cHH of Astacus leptodactylus carried out by solid phase peptide synthesis coupled to native chemical ligation. The synthetic 72 amino acid long peptide amides, containing L- or D-Phe(3) and (Glp(1), D-Phe(3)) were tested for their biological activity by means of homologous in vivo bioassays. The hyperglycemic activity of the D-isoforms was significantly higher than that of the L-isoform, while the presence of the N-terminal Glp residue had no influence on thepeptide activity. The results show that the presence of D-Phe(3) modifies the cHH functionality, contributing to the diversification of the hormone pool
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