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At least 1,099 records · Page 61Linked to original sources
PubMed2003

Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model.

Anthrax toxin is the dominant virulence factor of Bacillus anthracis; drugs blocking its action could therefore have therapeutic benefit. We report here the production of a neutralizing monoclonal antibody (mAb) against anthrax lethal factor (LF) and the inhibition by the antibody of anthrax lethal toxin (LeTx) formation. The anti-LF monoclonal antibody LF8 neutralized the LeTx challenge both in vitro with macrophage J774A.1 cells and in vivo in nude mice. Our data suggested that LF8 binds LF at or near the PA binding domain. A set of dodecameric peptides was selected from a phage-displayed peptide library through their specific binding to anti-LF neutralizing mAb LF8. These small peptides compete with LF to bind LF8. Further investigation is undergoing to test the potential application of these peptides to the clinical treatment of anthrax infection by blocking LeTx formation.

Animals↗
PubMed2000

Determining synthetic failures in combinatorial libraries by hybrid gas-phase separation methods.

A combinatorial tripeptide library having the general form D-Glu-Xxx-Xxx-CONH2 has been synthesized using a standard mix and split synthetic protocol that is expected to produce 676 components. All components of the mixture were analyzed using a new high-resolution ion mobility/time-of-flight mass spectrometer coupled with an electrospray ionization source. In this approach ions are separated by differences in their gas-phase mobilities prior to being introduced into the mass spectrometer for mass-to-charge analysis. The peptide library includes a wide range of different sequence, structural, and stereo isomers; trends in the number of expected and resolved isomers that are observed at each m/z ratio allow specific synthetic steps that have failed to be identified, even in the presence of other isomers. Information about the relative abundances of different isomers should dramatically improve the reliability of binding affinity studies from direct analysis of mixtures.

Chromatography, Gas↗
PubMed2005

Combinatorial approaches to functional models for galactose oxidase.

The copper enzyme galactose oxidase (GOase, EC 1.1.3.9) catalyses the oxidation of D-galactose and other primary alcohols in air to the corresponding aldehydes and hydrogen peroxide. The current mechanistic hypothesis for this two-electron redox reaction involves a Cu(I)/Cu(II) couple and the reversible oxidation of a ligating phenolate (tyrosine residue of the Tyr272-Cys228 conjugate) to a phenoxyl radical. Our approaches to functional models for galactose oxidase comprise both the use of low-molecular-weight copper complexes of a Schiff-base and sulfonamide ligands, and the synthesis/screening of combinatorial libraries. With regard to the latter, we have synthesized (by the IRORI-directed synthesis approach) peptide libraries carrying either His or the redox-active amino acids Tyr, mod-Cys (a model for the Tyr272-Cys228 conjugate) or TOAC (a TEMPO-derived alpha-amino acid) at four variable positions. After incubation with copper ions, the catalytically active library members were identified by specially designed screening methods.

Biomimetics↗
PubMed1999

Towards the pathogenesis of autoimmune liver disease.

There have been recent improvements in the clinical understanding and definition of the major types of autoimmune liver disease. However, still lacking is knowledge of their prevalence and pathogenesis. Three areas of study are in progress in our laboratory. First, in type 1 autoimmune hepatitis, the search continues to identify a liver/disease-specific autoantigenic reactant. Using hepatocyte membrane preparations, immunoblotting has underlined the problem of distinguishing, among multiple reactants, those that may be causally rather than consequentially related to hepatocellular damage. Second, in primary biliary cirrhosis (PBC), the need for population screening to ascertain prevalence and detect preclinical cases can be met by a rapid automated procedure for detection, by specific enzyme inhibition in microtitre wells, of antibody (anti-M2) to the pyruvate dehydrogenase complex E2 subunit (PDC-E2). Third, the structure of the conformational epitope within the inner lipoyl domain of PDC-E2 is being investigated by screening random phage-displayed peptide libraries using PBC sera. This has yielded phage clones in which the sequence of the peptide insert portrays the structure of this epitope, as judged by clustering of PBC-derived sequences to particular branches of a guide-tree that shows relatedness of peptides, and by reactivity of selected phage clones with anti-PDC-E2. Thus phage display identifies a peptide 'mimotope' of the antibody epitope in the inner lipoyl domain of PDC-E2.

Amino Acid Sequence↗
PubMed2001

Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage.

Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a "secondary" library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.

3T3 Cells↗
PubMed2005

Binding hot spot for invasion inhibitory molecules on Plasmodium falciparum apical membrane antigen 1.

Apical membrane antigen 1 (AMA1) is expressed in schizont-stage malaria parasites and sporozoites and is thought to be involved in the invasion of host red blood cells. AMA1 is an important vaccine candidate, as immunization with this antigen induces a protective immune response in rodent and monkey models of human malaria. Additionally, anti-AMA1 polyclonal and monoclonal antibodies inhibit parasite invasion in vitro. We have isolated a 20-residue peptide (R1) from a random peptide library that binds to native AMA1 as expressed by Plasmodium falciparum parasites. Binding of R1 peptide is dependent on AMA1 having the proper conformation, is strain specific, and results in the inhibition of merozoite invasion of host erythrocytes. The solution structure of R1, as determined by nuclear magnetic resonance spectroscopy, contains two structured regions, both involving turns, but the first region, encompassing residues 5 to 10, is hydrophobic and the second, at residues 13 to 17, is more polar. Several lines of evidence reveal that R1 targets a "hot spot" on the AMA1 surface that is also recognized by other peptides and monoclonal antibodies that have previously been shown to inhibit merozoite invasion. The functional consequence of binding to this region by a variety of molecules is the inhibition of merozoite invasion into host erythrocytes. The interaction between these peptides and AMA1 may further our understanding of the molecular mechanisms of invasion by identifying critical functional regions of AMA1 and aid in the development of novel antimalarial strategies.

Amino Acid Sequence↗
PubMed2001

[Studies on mimotopes of hepatitis C virus E1 protein].

OBJECTIVE: To study the B-cell epitope of E1 protein of hepatitis C virus. METHODS: By induction of IPTG, the E.coli M15 strains harboring the pQE30-HCVe118 expressed truncated C-terminal HCV E1 protein (Pte1). The proteins were purified with preparative electrophoreses system, which captured anti-E1 IgG in HCV (+) sera. By applying the antibodies as selective molecular 12 mers random peptide libraries were panned, and positive clones were obtained by ELISA. Amino acid sequences of display peptide were compared with that of HCV E1 protein. RESULTS: The purified HCV E1 proteins could react specifically with partly anti-HCV sera by ELISA. Among 10 phage display peptides, 6, 6, 2 were the most homologous to HCV E1 protein at position 320-336aa, 251-263aa, 225-248, respectively. CONCLUSIONS: There exist multiple B-cell epitopes in HCV E1 protein. At least one preponderant epitope is mapped at residues 320-336 of HCV E1 protein.

Amino Acid Motifs↗
PubMed2006

Peptide design of a competitive inhibitor for HMG-CoA reductase based on statin structure.

This study investigates a proposed design of a peptide sequence that is based on a bioactive conformation of statins that act as the competitive inhibitors of HMG-CoA for HMGR. To bridge these heterogeneous organic compounds, a conformational aspect relating to an analysis of the flexibility of the peptide molecules and their occupied volumes was applied to the peptide design. The design criterion was formulated in terms of a proximity parameter (Pr), reflecting the probability of an active peptide conformation to approximate the statin. Through a structure-functional analysis of previously synthesized peptides and statin molecules, nine peptides were selected for the peptide library. Comparing the calculated proximity parameters, four peptides (IAVE, YAVE, IVAE, and YVAE) from the library were selected and synthesized. In vitro assays elucidated the inhibition properties for HMGR that are exhibited by these peptides. Among all peptides, YVAE showed the highest ability to inhibit HMGR. A kinetic analysis revealed that this peptide is a competitive inhibitor of HMG-CoA with an equilibrium constant of inhibitor binding (K(i)) of 15.2 +/- 1.4 microM. The calculated coefficient correlation (R) between log (IC(50)) and the inverse value of proximity parameter (1/Pr) was found to be 0.99, indicating a high degree of correlation and efficacy of the given approach in the peptide sequence design.

Amino Acid Sequence↗
PubMed2001

The N-terminal domain of Homer/Vesl is a new class II EVH1 domain.

Cellular activities controlled by signal transduction processes such as cell motility and cell growth depend on the tightly regulated assembly of multiprotein complexes. Adapter proteins that specifically interact with their target proteins are key components required for the formation of these assemblies. Ena/VASP-homology 1 (EVH1) domains are small constituents of large modular proteins involved in microfilament assembly that specifically recognize proline-rich regions. EVH1 domain-containing proteins are present in neuronal cells, like the Homer/Vesl protein family that is involved in memory-generating processes. Here, we describe the crystal structure of the murine EVH1 domain of Vesl 2 at 2.2 A resolution. The small globular protein consists of a seven-stranded antiparallel beta-barrel with a C-terminal alpha-helix packing alongside the barrel. A shallow groove running parallel with beta-strand VI forms an extended peptide-binding site. Using peptide library screenings, we present data that demonstrate the high affinity of the Vesl 2 EVH1 domain towards peptide sequences containing a proline-rich core sequence (PPSPF) that requires additional charged amino acid residues on either side for specific binding. Our functional data, substantiated by structural data, demonstrate that the ligand-binding of the Vesl EVH1 domain differs from the interaction characteristics of the previously examined EVH1 domains of the Evl/Mena proteins. Analogous to the Src homology 3 (SH3) domains that bind their cognate ligands in two distinct directions, we therefore propose the existence of two distinct classes of EVH1 domains.

Amino Acid Sequence↗
PubMed2004

Mimotopes of conformational epitopes in fibrillar beta-amyloid.

In Alzheimer's disease (AD) beta-amyloid peptide accumulates in the brain in different forms including fibrils. Amyloid fibrils could be recognized as foreign by the mature immune system since they are not present during its development. Thus, using mouse antisera raised against the fibrillar form of Abeta42, we have screened two phage peptide libraries for the presence of foreign conformational mimotopes of Abeta. Antisera from wild type animals recognized predominately peptides with the EFRH motif from Abeta42 sequence, whereas amyloid precursor protein (APP) transgenic mice recognized mainly phage clones that mimic epitopes (mimotopes) within the fibrillar Abeta42 but lack sequence homology with this peptide.

Amino Acid Sequence↗
PubMed2003

Inhibition of Staphylococcus aureus pathogenesis in vitro and in vivo by RAP-binding peptides.

Staphylococcus aureus cause many diseases by producing toxins, whose synthesis is regulated by quorum-sensing mechanisms. S. aureus secretes a protein termed RNAIII activating protein (RAP) which autoinduces toxin production via the phosphorylation of is target protein TRAP. Mice vaccinated with RAP were protected from S. aureus infection, suggesting that RAP is an useful target for selecting potential therapeutic molecules to inhibit S. aureus pathogenesis. We show here that RAP (native and recombinant) was used to select RAP-binding peptides (RBPs) from a random 12-mer phage-displayed peptide library. Two RBPs were shown to inhibit RNAIII production in vitro (used a marker for pathogenesis). The peptide WPFAHWPWQYPR, which had the strongest inhibitory activity, was chemically synthesized and also expressed in Escherichia coli as a GST-fusion. Both synthetic peptide and GST-fusion peptide decreased RNAIII levels in a dose-dependent manner. The GST-fusion peptide was also shown to protect mice from a S. aureus infection in vivo (tested in a murine cutaneous S. aureus infection model). Our results suggest the potential use of RAP-binding proteins in treating clinical S. aureus infections.

Animals↗
PubMed2002

Phage-displayed peptides bind to the malarial protein apical membrane antigen-1 and inhibit the merozoite invasion of host erythrocytes.

Apical membrane antigen-1 (AMA1) is a transmembrane protein present on the surface of merozoites that is thought to be involved in the process of parasite invasion of host erythrocytes. Although it is the target of a natural immune response that can inhibit invasion, little is known about the molecular mechanisms by which AMA1 facilitates the invasion process. In an attempt to identify peptides that specifically interact with and block the function of AMA1, a random peptide library displayed on the surface of filamentous phage was panned on recombinant AMA1 from Plasmodium falciparum. Three peptides with affinity for AMA1 were isolated, and characterization of their fine binding specificities indicated that they bind to a similar region on the surface of AMA1. One of these peptides was found to be a potent inhibitor of the invasion of P. falciparum merozoites into human erythrocytes. We propose that this peptide blocks interaction between AMA1 and a ligand on the erythrocyte surface that is involved in a critical step in malarial invasion. The identification and characterization of these peptide inhibitors now permit an evaluation of the essential requirements that are necessary for efficient neutralization of merozoite invasion by blocking AMA1 function.

Alanine↗
PubMed2001

Designing a polyvalent inhibitor of anthrax toxin.

Screening peptide libraries is a proven strategy for identifying inhibitors of protein-ligand interactions. Compounds identified in these screens often bind to their targets with low affinities. When the target protein is present at a high density on the surface of cells or other biological surfaces, it is sometimes possible to increase the biological activity of a weakly binding ligand by presenting multiple copies of it on the same molecule. We isolated a peptide from a phage display library that binds weakly to the heptameric cell-binding subunit of anthrax toxin and prevents the interaction between cell-binding and enzymatic moieties. A molecule consisting of multiple copies of this nonnatural peptide, covalently linked to a flexible backbone, prevented assembly of the toxin complex in vitro and blocked toxin action in an animal model. This result demonstrates that protein-protein interactions can be inhibited by a synthetic, polymeric, polyvalent inhibitor in vivo.

Acrylic Resins↗
PubMed1998

Synthesis and hybridization analysis of a small library of peptide-oligonucleotide conjugates.

A small library of 49 peptide-oligonucleotide conjugates were synthesized to explore the influence of various peptide side chains on the hybridization properties of the DNA. An invariant 8mer oligonucleotide was coupled to a peptide portion that contained a five residue variable region composed of the cationic amino acids lysine, ornithine, histidine and arginine, the hydrophobic amino acid tryptophan, and alanine as a spacer. Melting temperature analysis indicated that T m depended principally on the number of cationic residues. The free energies of binding for polycationic peptide-oligonucleotides were enhanced compared with the unmodified 8mer. The origin of this stabilizing effect was found to be derived from a more exothermic enthalpic term. Improvement in Delta G vH was found to depend on the presence of positive charge and also the exact identity of the cationic amino acid, with the polyarginine peptide giving the most favourable Delta G vH value and the most exothermic Delta H vH. Further exploration suggested that the cationic peptide fragments interacted mainly with single-stranded rather than duplex DNA. A study of pH dependence showed that the polyhistidine conjugate was particularly sensitive to pH changes near neutrality, as indicated by a significant rise in T m from 19.5 degrees C at pH 8.0 to 28.5 degrees C at pH 6.0.

Chromatography, High Pressure Liquid↗
PubMed2005

Localized transfection on arrays of magnetic beads coated with PCR products.

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.

Animals↗
PubMed1997

Cell-surface interactions of echovirus 22.

Echovirus 22 (EV22) is a picornavirus forming a distinct molecular cluster together with echovirus 23. EV22 has an Arg-Gly-Asp (RGD) peptide motif in its capsid protein VP1; similar motifs are known to mediate many cell-cell and microbe-host interactions. To identify peptide sequences that specifically bind to EV22 and potentially play a role in receptor recognition, we have used here peptide libraries displayed in filamentous phage. We isolated an EV22-binding motif CLRSG(R/F)GC. The synthetic CLRSGRGC peptide was able to inhibit EV22 infection. The infection was also inhibited by an RGD-containing peptide representing the C terminus of the EV22 capsid protein VP1 and CWDDGWLC (an RGD-binding peptide; Pasqualini, R., Koivunen, E., and Ruoslahti, E. (1995) J. Cell Biol. 130, 1189-1196). As the EV22-recognizing sequence LRSG is found in the integrin beta1 chain and the entire LRSGRG hexapeptide occurs in the matrix metalloproteinase 9 (MMP-9), we carried out blocking experiments with anti-integrin and anti-MMP-9 antibodies. EV22 infection could be blocked in cell cultures with anti-alphav, -beta1, and, to a lesser extent, with anti-MMP-9 antibodies. These results imply that EV22 recognizes preferentially alphavbeta1-integrin as a cellular receptor and MMP-9 may also play a role in the cell-surface interactions of the virus.

Amino Acid Sequence↗
PubMed1998

Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains.

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX1-2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Generation and utilization of synthetic combinatorial libraries.

The use of combinatorial chemistry is fundamentally changing the pace and scope of basic research and drug discovery. Since the introduction of synthetic peptide libraries several years ago, combinatorial chemistry has proven to be a powerful tool for the generation of immense molecular diversities of peptides, peptidomimetics and new organic compounds. This article briefly reviews methods for the generation and application of combinatorial libraries, with particular emphasis on soluble synthetic combinatorial libraries. The utility of these molecular diversities for basic research and drug discovery has been demonstrated through the identification of numerous highly active compounds such as antigenic peptides, receptor ligands, antimicrobial compounds and enzyme inhibitors.

Amino Acid Sequence↗