The Design and Construction of Synthetic Protein Mimics

A strategy for the modular construction of synthetic protein mimics based on the ability non-protein amino acids to act as stereochemical directors of polypeptide chain folding, is described. The use of alpha-aminoisobutyric acid (Aib) to construct stereochemically rigid helices has been exemplified by crystallographic and spectroscopic studies of several apolar peptides, ranging in length from seven to sixteen residues. The problem of linker design in elaborating alpha,alpha motifs has been considered. Analysis of protein crystal structure data provides a guide to choosing linking sequences. Attempts at constructing linked helical motifs using linking Gly-Pro segments have been described. The use of flexible linkers, like epsilon-aminocaproic acid has been examined and the crystallographic and solution state analysis of a linked helix motif has been presented. The use of bulky sidechain modifications on a helical scaffold, as a means of generating putative binding sites has been exemplified by a crystal structure of a peptide packed in a parallel zipper arrangement.

Molecular Design of Synthetic Benzimidazoles for the Switchover of the Duplex to G-quadruplex DNA Recognition

Benzimidazole derivatives are well known for their antibacterial, antiviral, anticonvulsant, antihistaminic, anthelmintic and antidepressant activities. Benzimidazole's unique base-selective DNA recognition property has been studied widely. However, most of the early benzimidazole systems have been targeted towards the binding of duplex DNA. Here we have shown the evolution and progress of the design and synthesis of new benzimidazole systems towards selective recognition of the double-stranded DNA first. Then in order to achieve selective recognition of the G-quadruplex DNA and utilize their potential as future anti-cancer drug candidates, we have demonstrated their selective cytotoxicity towards the cancer cells and potent telomerase inhibition ability.

A cyclic peptide mimic of an RNA recognition motif of human La protein is a potent inhibitor of hepatitis C virus

Due to limited available therapeutic options, developing new lead compounds against hepatitis C virus is an urgent need. Human La protein stimulates hepatitis C virus translation through interaction with the hepatitis C viral RNA. A cyclic peptide mimicking the beta-turn of the human La protein that interacts with the viral RNA was synthesized. It inhibits hepatitis C viral RNA translation significantly better than the corresponding linear peptide at longer post-treatment times. The cyclic peptide also inhibited replication as measured by replicon RNA levels using real time RT-PCR. The cyclic peptide emerges as a promising lead compound against hepatitis C.

Multivalent glycoliposomes and micelles to study carbohydrate-protein and carbohydrate-carbohydrate interactions

This tutorial review describes multivalent carbohydrate-protein and carbohydrate-carbohydrate interaction studies that utilize self-assembled aggregates of thermodynamically stable liposomes and micelles. Strategies to prepare multivalent glycoliposomes and micelles include: (i) insertion of synthetic glycolipids into matrix lipids; (ii) preparation of glycolipids that aggregate to liposomes and micelles and (iii) modification of the hydrophilic surfaces with desired sugars. Several design strategies have been developed in order to obtain constituent glycolipids, having multivalent sugar moieties and their subsequent interactions with proteins were assessed in relation to the type of linkers that connect the hydrophilic and lipophilic segments. Lipophilic segments other than alkyl chains have also been developed. Polymer based glycoliposomes and micelles form an emphasis. Further, glycoliposomes facilitate studies of carbohydrate-carbohydrate interactions. An overview of the various types of glycoliposomes and micelles used to study carbohydrate-protein and carbohydrate-carbohydrate recognition phenomena is presented.

An Overview on the Field of Micro- and Nanotechnologies for Synthetic Peptide-Based Vaccines

18 p.

Studies of Organic Molecule Recognition by Synthetic Cyclophane Receptors in Aqueous Media

The behaviors of six new cyclophane receptors for organic guest molecules in aqueous media are reported. These new hosts are modifications of more basic parent structures, and the main goal of their examination has been to determine how the modifications affect host selectivity for cationic guests. In particular, we have been interested in determining how additional non-covalent binding interactions can complement the cation-π interactions active in the parent systems. Three types of modifications were made to these systems. Firstly, neutral methoxy and bromine substituents were added to produce four of the six new macrocycles. Secondly, two additional aromatic rings (relative to the parent host) capable of making cation-π interactions with charged guest species were appended. Thirdly, a negatively charged carboxyl group was attached to produce a cavity in which electrostatic interactions should enhance cationic guest binding. ^1H-NMR and circular dichroic techniques were employed to determine the binding affinities of a wide variety of organic guests for the parent and modified structures in aqueous media.

Bromination of the parent host greatly enhances its binding in a general fashion, primarily as the result of hydrophobic interactions. The addition of methoxy groups does not enhance binding, apparently as a result of a collapse of the hosts into a conformation that is not suitable for binding. The appendage of extra aromatic rings enhances the binding of positively charged guests, most likely in response to more complete encapsulation of guest species. The addition of a negatively charged carboxylate enhances the binding to only selective groups of cationic guests. AM1 calculations of the electrostatic potentials of several guests molecules suggests that the enhancements seen with the modified receptor compared to the parent are most likely the result of close contact between regions of highest potential on the guest and the appended carboxylate.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated Suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. (C) 2008 Elsevier B.V. All rights reserved.

Immune Responses to Six Synthetic Peptides of Capsid Protein Immune Responses to Six Synthetic Peptides of Capsid Protein

Many B cell epitopes within p24 of human immunodeficiency virus type 1 (HIV-1) were identified, while most of them were determined by using murine monoclonal antibodies reacting with overlapping peptides of p24. Therefore these epitopes may not represent the actual epitopes recognized by the HIV-1 infected individuals. In the present study, immune responses of 67 HIV-1 positive sera from Yunnan Province, China to five peptides on p24 of HIV-1 and one of HIV-2 were analyzed. All of 67 sera did not recognize peptide GA-12 on HIV-1 and peptide AG-23 on HIV-2, which indicated that GA-12 was not human B cell epitope and AG-23 did not cross-react with HIV-1 positive serum. Except 13 sera (19.4%), all remaining sera did not recognize peptides NI-15, DR-16, DC-22 and PS-18, which indicated that these four peptides represented B cell linear epitopes of HIV-1 p24 in some HIV-1 infected individuals but not the immuno-dominant epitopes in most individuals. Cellular & Molecular Immunology. 2005;2(4):289-293.

The expression of peptidoglycan recognition protein-S1 gene in the scallop Chlamys farreri was enhanced after a second challenge by Listonella anguillarum

A more rapid and powerful response against repeated exposure of same pathogen in vertebrates is usually considered as the reflection of immunological memory, but it is not well understood in invertebrates. In the present Study, the temporal expression profiles of Chlamys farreri peptidoglycan recognition protein-S1 (CfPGRP-S1) gene after two challenges of Listonella anguillarum were examined to evaluate priming response in scallops. The up-regulation of CFPGRP-S1 mRNA occurred 3 h earlier, and the expression level was significant higher (P < 0.05), after the second challenge than that after the first challenge. The preliminary results Provided new insights into invertebrate immunological memory, and they also would be helpful to develop strategies for disease control. (C) 2008 Elsevier Inc. All rights reserved.

Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis

A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp.

A fibrinogen-related protein from bay scallop Argopecten irradians involved in innate immunity as pattern recognition receptor

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like domains. Many members of this family play important roles as pattern recognition receptors in innate immune responses. The cDNA of bay scallop Argopecten irradians FREP (designated as AiFREP) was cloned by rapid amplification of cDNA ends (RACE) method based on the expressed sequence tag (EST). The full-length cDNA of AiFREP was of 990 bp. The open reading frame encoded a polypeptide of 251 amino acids, including a signal sequence and a 213 amino acids fibrinogen-like domain. The fibrinogen-like domain of AiFREP was highly similar to those of mammalian ficolins and other FREPs. The temporal expression of AiFREP mRNA in hemolymph was examined by fluorescent quantitative real-time PCR. The mRNA level of scallops challenged by Listonella anguillarum was significantly up-regulated, peaked to 9.39-fold at 9 h after stimulation, then dropped back to 4.37-fold at 12 h, while there was no significant change in the Micrococcus luteus challenged group in all periods of treatment. The function of AiFREP was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiFREP (rAiFREP) agglutinated chicken erythrocytes and human A, B, O-type erythrocytes. The agglutinating activities were calcium-dependent and could be inhibited by acetyl group-containing carbohydrates. rAiFREP also agglutinated Gram-negative bacteria E. coli JM109, L anguillarum and Gram-positive bacteria M. luteus in the presence of calcium ions. These results collectively suggested that AiFREP functions as a pattern recognition receptor in the immune response of bay scallop and contributed to nonself recognition in invertebrates, which would also provide clues for elucidating the evolution of the lectin pathway of the complement system. (C) 2008 Elsevier Ltd. All rights reserved.