Publication date:
Author(s): Nelson Marmiroli
Publication date:
Author(s): Nelson Marmiroli
Members of the Chordopoxvirinae subfamily possess an unusual 11 protein entry–fusion complex (EFC) that is highly conserved and present in all species. The mode of action of this EFC is unknown, and the interactions of the constituent proteins are uncharacterised. Here, we present the chemical synthesis of membrane domain truncated linear constructs of two EFC proteins in orf virus, ORFV036 and 049. By using Boc solid phase peptide synthesis and native chemical ligation methods, these truncated proteins have been readily prepared in milligram quantities. These robust synthetic protocols allow ready access to these polypeptides to facilitate biological studies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Orf virus requires an unusually large ≥11-protein entry fusion complex to enter host cells. Through chemical synthesis, we have prepared truncated constructs of two of these entry fusion proteins (ORFV036 and 049), which were previously found to be intractable to bacterial expression. The robust synthetic protocols used allow access to milligram quantities in high purity for further biological studies.
The indiscriminate usage of antibiotics has created a major problem in the form of antibiotic resistance. Even though new antimicrobial drug discovery programs have been in place from the last two decades, still we are unsuccessful in identifying novel molecules that have a potential to become new therapeutic agents for the treatment of microbial infections. A major problem in most screening studies is the requirement of high-throughput techniques. Given this, we present here an enzyme-based robust method for screening antimicrobial agent's active against Escherichia coli. This method is based upon the ability of the intracellular innate enzyme to cleave o-nitrophenyl β-d-galactopyranoside (non-chromogenic) to o-nitrophenolate (ONP) (chromogenic) upon the membrane damage or disruption. In comparison with the other currently available methods, we believe that our method provides an opportunity for real-time monitoring of the antimicrobial agents action by measuring the ONP generation in a user-friendly manner. Even though this method can be applied to other strain, our experience shows that one has to be careful especially when the pigments or metabolites present in the bacteria have the same wavelength absorbance. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Antimicrobial peptides (AMPs) are a critical component of the natural defense system with multifunctional roles. Recently, there is an increased interest as they are explored as an alternative to conventional antibiotics. Here, we present a simple, robust, cost-effective enzyme-based method for the discovery of novel antimicrobial agents with real-time monitoring of the antimicrobial agents action, in addition to the ability to test the AMPs in near physiological conditions.
We show that three designed cyclic d,l-peptides are β-helical in TFE—a solvent in which the archetypal β-helical peptide, gA, is unstructured. This result represents an advance in the field of β-helical peptide foldamers and a step toward achieving β-helical structure under a broad range of solvent conditions. We synthesized two of the three peptides examined using an improved variant of our original CBC strategy. Here, we began with a commercially available PEG–PS composite resin prefunctionalized with the alkanesulfonamide ‘SCL’ linker and preloaded with glycine. Our new conditions avoided C-terminal epimerization during the CBC step and simplified purification. In addition, we present results to define the scope and limitations of our CBC strategy. These methods and observations will prove useful in designing additional cyclic β-helical peptides for applications ranging from transmembrane ion channels to ligands for macromolecular targets. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Resin-bound linear d,l-peptides of representative sequence lin-WS β undergo racemization-free CBC. The resulting cyclic d,l-peptides (e.g. WS β) are highly β-helical in TFE – a solvent in which the archetypal β-helical peptide, gA, is unstructured. These findings represent a step toward designing β-helical structures for applications in a variety of solvent conditions.
Two glycosylated peptides have been studied using NMR spectroscopy supported by molecular modeling. Peptide I is an oxytocin (OT) analogue in which glutamine 4 was replaced by serine with attached α-d-mannose through the oxygen β atom, whereas peptide II is a lysine-vasopressin (LVP) analogue with lysine 8 side chain modified by the attachment of glucuronic acid through an amide bond. Both peptides exhibit very weak uterotonic effect and are less susceptible to proteolytic degradation than the mother hormones. Additionally, peptide II reveals very weak pressor and antidiuretic activities. Our results have shown that the conformational preferences of glycosylated analogues are highly similar to those of their respective mother hormones. OT glycosylated analogue (I) exhibits a 3,4 β-turn characteristic of OT-like peptides, and vasopressin-glycosylated analogue (II) exhibits β-turns typical of vasopressin-like peptides. Therefore, the lack of binding of the glycosylated analogues to the receptors can be attributed to a steric interference between the carbohydrate moieties and the receptors. We also consider this to be the reason of the very low activity of the analyzed glycopeptides. We expect that results from these studies will be helpful in designing new OT-like and vasopressin-like drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
The introduction of the sterically restricted bulky glycoconjugates significantly reduces the activity of the neurohypophyseal hormones analogues. We consider that the reason of this can be steric repulsion between the carbohydrate moiety and the receptors. Therefore, despite that oxytocin-glycosylated analogue I exhibits 3,4 β-turn characteristic of oxytocin-like peptides and vasopressin-glycosylated analogue II exhibits β-turns typical of vasopressin-like peptides, these analogues have very low activity.
Publication date:
Author(s): Kairong Wang , Jiexi Yan , Wen Dang , Junqiu Xie , Bo Yan , Wenjin Yan , Mengyang Sun , Bangzhi Zhang , Mingxia Ma , Yanyan Zhao , Fengjing Jia , Ranran Zhu , Wei Chen , Rui Wang
A novel antimicrobial peptide, designated macropin (MAC-1) with sequence Gly-Phe-Gly-Met-Ala-Leu-Lys-Leu-Leu-Lys-Lys-Val-Leu-NH2, was isolated from the venom of the solitary bee Macropis fulvipes. MAC-1 exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l- or d-lysine in selected positions. Furthermore, all-d analog and analogs with d-amino acid residues introduced at the N-terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α-helical secondary structure in the presence of trifluoroethanol or membrane-mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d-amino acid substitutions in MAC-1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Novel peptide named macropin was isolated from the venom of wild bee Macropis fulvipes. It belongs to the category of amphipathic α-helical antimicrobial peptides as illustrated by its wheel projection. A series of macropin analogs were prepared to evaluate the effect of structural alterations on biological activity. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were increased while their toxicity against red blood cells was decreased. The structure of macropin and selected analogs was elucidated by NMR.
Vicinal disulfide rings (VDRs) occur when a disulfide bond forms between adjacent cysteine residues in a protein and results in a rare eight-membered ring structure. This eight-membered ring has been found to exist in four major conformations in solution, divided between cis and trans conformers. Some selenoenzymes use a special type of VDR in which selenium replaces sulfur, generating a vicinal selenosulfide ring (VSeSR). Here, we provide evidence that this substitution reduces ring strain, resulting in a strong preference for the trans conformation relative to cis in a VSeSR (cis:trans – 9:91). This was determined by using the ‘γ-gauche effect’, which makes use of both 1H-NMR and two-dimensional (2D) NMR techniques for determining the amide bond conformeric ratio. The presence of selenium in a VSeSR also lowers the dihedral strain energy (DSE) of the selenosulfide bond relative to the disulfide bond of VDRs. While cis amide geometry decreases strain on the amide bond, it increases strain on the scissile disulfide bond of the VDR found in thioredoxin reductase from Drosophila melanogaster (DmTR). We hypothesize that the cis conformation of the VDR is the catalytically competent conformer for thiol/disulfide exchange. This hypothesis was investigated by computing the DSE of VDR and VSeSR conformers, the structure of which was determined by 2D NMR spectroscopy and energy minimization. The computed values of the VDR from DmTR are 16.5 kJ/mol DSE and 14.3 kJ/mol for the C+ and T− conformers, respectively, supporting the hypothesis that the enzyme uses the C+ conformer for thiol/disulfide exchange. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Some enzymes use a rare vicinal disulfide ring for catalysis. These disulfide rings are strained and may fluctuate in the enzyme between cis and trans amide conformations. We show that the cis conformer may be important for thiol/disulfide exchange. Mammalian thioredoxin reductase uses such a motif, except that one sulfur atom of the ring is replaced with selenium. The consequence of this substitution is that the trans form of the ring is stabilized and is the predominant conformer in solution.
While genome-wide association studies as well as candidate gene studies have revealed a great deal of insight into the contribution of genetics to obesity development and susceptibility, advances in adipose tissue research have substantially changed the understanding of adipose tissue function. Its perception has changed from passive lipid storage tissue to active endocrine organ regulating and modulating whole-body energy homeostasis and metabolism and inflammatory and immune responses by secreting a multitude of bioactive molecules, termed adipokines.
The expression of human vaspin (serpinA12) is positively correlated to body mass index and insulin sensitivity and increases glucose tolerance in vivo, suggesting a compensatory role in response to diminished insulin signaling in obesity. Recently, considerable insight has been gained into vaspin structure, function, and specific target tissue-dependent effects, and several lines of evidence suggest vaspin as a promising candidate for drug development for the treatment of obesity-related insulin resistance and inflammation. These will be summarized in this review with a focus on molecular mechanisms and pathways. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Considerable insight has been gained into vaspin structure, function, and specific target tissue-dependent effects, and several lines of evidence suggest vaspin as a promising candidate for drug development for the treatment of obesity-related insulin resistance and inflammation. This review will summarize recent findings with a focus on molecular mechanisms and pathways.
O-Acyl isopeptides, in which the N-acyl linkage on the hydroxyamino acid residue (e.g. Ser and Thr) is replaced by an O-acyl linkage, generally suppress unfavorable aggregation properties derived from the corresponding parent peptides. Here, we report the synthesis of an O-acyl isopeptide of 34-mer pyroGlu-ADan (2), a component of amyloid deposits in hereditary familial Danish dementia, by using native chemical ligation. Native chemical ligation of pyroGlu1-ADan(1-21)-SCH2CH2SO3−Na+ (3) and Cys22-O-acyl isopeptide (4), in which the amino group of the Ser29 residue at the isopeptide moiety was protected by an allyloxycarbonyl group, proceeded well in an aqueous solvent to yield a ligated O-acyl isopeptide (5). Subsequent disulfide bond formation and deprotection of the allyloxycarbonyl group followed by HPLC purification gave 2 with a reasonable overall yield. 2 was converted to the parent peptide 1 via an O-to-N acyl migration reaction. The sequential method, namely (i) native chemical ligation of the O-acyl isopeptide, (ii) HPLC purification as the O-acyl isopeptide form, and (iii) O-to-N acyl migration into the desired polypeptide, would be helpful to solve problems with HPLC purification of hydrophobic polypeptides in the process of chemical protein synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
We here reported the synthesis of an O-acyl isopeptide by using native chemical ligation in an aqueous solvent, in combination with orthogonal allyloxycarbonyl protecting group at the amino group of the isopeptide moiety. The O-acyl isopeptide was quantitatively converted to the parent native peptide via an O-to-N acyl migration reaction. The present method would be helpful to solve the problem of HPLC purification of hydrophobic polypeptides in the process of chemical protein synthesis by using ligation chemistry.