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PubMedDate not supplied

T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein.

Proteins containing tandemly repetitive sequences are present in several immunodominant protein antigens in pathogenic protozoan parasites. The tandemly repetitive Trypanosoma cruzi B13 protein is recognized by IgG antibodies from 98% of Chagas' disease patients. Little is known about the molecular mechanisms that lead to the immunodominance of the repeated sequences, and there is limited information on T cell epitopes in such repetitive antigens. We finely characterized the T cell recognition of the tandemly repetitive, degenerate B13 protein by T cell lines, clones and PBMC from Chagas' disease cardiomyopathy (CCC), asymptomatic T. cruzi infected (ASY) and non-infected individuals (N). PBMC proliferative responses to recombinant B13 protein were restricted to individuals bearing HLA-DQA1*0501(DQ7), -DR1, and -DR2; B13 peptides bound to the same HLA molecules in binding assays. The HLA-DQ7-restricted minimal T cell epitope [FGQAAAG(D/E)KP] was identified with an overlapping combinatorial peptide library including all B13 sequence variants in T. cruzi Y strain B13 protein; the underlined small residues GQA were the major HLA contact residues. Among natural B13 15-mer variant peptides, molecular modeling showed that several variant positions were solvent (TCR)-exposed, and substitutions at exposed positions abolished recognition. While natural B13 variant peptide S15.9 seems to be the immunodominant epitope for Chagas' disease patients, S15.4 was preferentially recognized by CCC rather than ASY patients, which may be pathogenically relevant. This is the first thorough characterization of T cell epitopes of a tandemly repetitive protozoan antigen and may suggest a role for T cell help in the immunodominance of protozoan repetitive antigens.

Amino Acid Sequence↗
PubMed2006

Tailoring surface properties to build colloidal diagnostic devices: controlling interparticle associations.

The ability to control the surface properties and subsequent colloidal stability of dispersed particles has widespread applicability in many fields. Sub-micrometer fluorescent silica particles (reporters) can be used to actively encode the combinatorial synthesis of peptide libraries through interparticle association. To achieve these associations, the surface chemistry of the small fluorescent silica reporters is tailored to encourage robust adhesion to large silica microparticles onto which the peptides are synthesized. The interparticle association must withstand a harsh solvent environment, multiple synthetic and washing procedures, and biological screening buffers. The encoded support beads were exposed to different solvents used for peptide synthesis, and different solutions used for biological screening including phosphate buffered saline (PBS), 2-[N-morpholino]ethane sulfonic acid (MES) and a mixture of MES and N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide (EDC). The number of reporters remaining adhered to the support bead was quantified after each step. The nature of the associations were explored and tested to optimize the efficiency of these phenomena. Results presented illustrate the influence of the surface functionality and polyelectrolyte modification of the reporters. These parameters were investigated through zeta potential and X-ray photoelectron spectroscopy.

Journal Article↗
PubMed1997

Identification of a surrogate TSL-1 antigen of Trichinella spiralis from a phage library of random peptides.

We sought to find a peptide analogue of an important antigen (TSL-1) of Trichinella spiralis which is recognized by the 7C2C5 antibody. A phage library which displays a short (15-mer) randomly-generated peptide at the filament of the minor coat protein of the virion was used for selection by the 7C2C5 antibody. A peptide thus identified, ICDASGLGCWCWSLSP, was found to be a true surrogate since its binding to the antibody could be blocked by the native antigen and, conversely, an antiserum made to the peptide could recognize the native antigen. In addition, the peptide appeared to detect T. spiralis-infected pigs although it was less discriminatory than the native antigen.

Amino Acid Sequence↗
PubMed2000

Differential sensitivity to mutations in a single peptide by two TCRs having identical beta-chains and closely related alpha-chains.

The TCR on CD4 T cells binds to and recognizes MHC class II:antigenic peptide complexes through molecular contacts with the peptide amino acid residues that face up and out of the peptide-binding groove. This interaction primarily involves the complementarity-determining regions (CDR) of the TCR alpha- and ss-chains contacting up to five residues of the peptide. We have used two TCRs that recognize the same antigenic peptide and have identical Vss8.2 chains, but differ in all three CDR of their related Valpha2 chains, to examine the fine specificity of the TCR:peptide contacts that lead to activation. By generating a peptide library containing all 20 aa residues in the five potential TCR contact sites, we were able to demonstrate that the two similar TCRs responded differentially when agonist, nonagonist, and antagonist peptide functions were examined. Dual substituted peptides containing an agonist residue at the N terminus, which interacts with CDR2alpha, and an antagonist residue at the C terminus, which interacts with the CDR3ss, were used to show that the nature of the overall signal through the TCR is determined by a combination of the type of signal received through both the TCR alpha- and ss-chains.

Amino Acid Sequence↗
PubMed1996

Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries.

Ideal gene therapy vectors would be delivered intravenously to transfect only specific cells. Existing vectors only transfect cells in vivo in a manner determined by blood flow and the site of introduction. As a general and systematic approach for generating cell-targeting ligands for gene therapy vectors, we have used peptide-presenting phage libraries to select peptides that bind and enter several different cell types. Because of their small size, cell-binding peptides such as these could be incorporated into biological or physical gene therapy vectors. In addition, peptide-presenting phage themselves may also be candidates for gene therapy vectors.

Amino Acid Sequence↗
PubMed1999

A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry.

A method has been developed for de novo peptide sequencing using matrix-assisted laser desorption ionization mass spectrometry. This method will facilitate biological studies that require rapid determination of peptide or protein sequences, e.g., determination of posttranslational modifications, identification of active compounds isolated from combinatorial peptide libraries, and the selective identification of proteins as part of proteome studies. The method involves fast, one-step addition of a sulfonic acid group to the N terminus of tryptic peptides followed by acquisition of postsource decay (PSD) fragment ion spectra. The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions). The overall method has been applied to pmol quantities of peptides. The resulting PSD fragment ion spectra often exhibit uninterrupted sequences of 20 or more amino acid residues. However, fragmentation efficiency decreases considerably at amide bonds on the C-terminal side of Pro. The spectra are simple enough that de novo sequence tagging is routine. The technique has been successfully applied to peptide mixtures, to high-mass peptides (up to 3,600 Da) and to the unambiguous identification of proteins isolated from two-dimensional gel electrophoresis. The PSD spectra of these derivatized peptides often allow far more selective protein sequence database searches than those obtained from the spectra of native peptides.

Amino Acid Sequence↗
PubMed2005

Pathogenic T-cell clones in autoimmune diabetes: more lessons from the NOD mouse.

T-cell clones that can efficiently transfer diabetes to prediabetic nonobese diabetic (NOD) mice provide a powerful approach to dissecting the autoimmune disease process and for investigating immunoregulation. Diabetogenic T-cell clones carried in culture allow for detailed analysis of T-cell effector function and in vivo activity, and thus the contribution of a single clonotype to pathogenesis can be studied. As T cells comprising most or all of the repertoire in T-cell receptor transgenic (TCR-Tg) mice, diabetogenic T-cell clones have led to new variations on the NOD mouse model of autoimmune disease. T-cell clones are being used to screen peptide libraries and proteomic arrays to identify the autoantigens that drive these clones in vivo and to extend our knowledge of the processes that give rise to these antigens. With the identification of peptide agonists and natural ligands, the development of MHC-peptide multimers has been possible. These reagents can track T cells in vivo and thus provide new approaches for disease diagnosis and therapy as well as a versatile set of tools for basic research on how T cells contribute to autoimmune disease.

Animals↗
PubMed1996

The multiple endocrine neoplasia type 2B point mutation alters long-term regulation and enhances the transforming capacity of the epidermal growth factor receptor.

The RET proto-oncogene encodes a member of the receptor tyrosine kinase family. Multiple endocrine neoplasia type 2B (MEN 2B) is caused by the mutation of a conserved methionine to a threonine in the catalytic domain of the RET kinase. When the MEN 2B point mutation was introduced into the epidermal growth factor (EGF) receptor (M857T EGFR), the intrinsic tyrosine kinase activity of the mutant receptor was similar to that of wild-type EGF receptor and remained ligand-dependent. However, the mutant receptor showed an enhanced transforming capacity compared to the wild-type receptor as judged by its ability to mediate the growth of NIH 3T3 cells in soft agar. Using the oriented peptide library approach to examine substrate specificity, the M857T mutation was found to be associated with a decrease in the selectivity of the receptor for Phe and an increase in the selectivity for acidic residues at the P + 1 position as compared to wild-type EGF receptor. Short-term responses to EGF were similar in cells expressing wild-type and M857T EGF receptors. However, significant differences in receptor down-regulation were observed between the two receptors. These data demonstrate that the MEN 2B point mutation alters the substrate specificity of receptor tyrosine kinases and suggest that the enhanced oncogenesis associated with the MEN 2B mutation may be due in part to alterations in receptor regulation.

3T3 Cells↗
PubMed2002

An approach to increased polyplex gene delivery by peptides selected from a phage display library.

Phage display libraries were screened for peptides to be incorporated in nonviral gene delivery vehicles. Cells in culture were incubated with heptamer random peptide libraries displayed on M13 bacteriophages in three to five copies per phage. Surface-adherent phages were removed or inactivated and the cells were fractionated in a nuclear pellet and supernatant. Bacteriophages from each of the two fractions were amplified and reincubated with the cells. Three successive rounds of selection were performed. Eighteen sequenced clones revealed 14 different sequences. Two sequences were homologous to segments of the HIV gp120 protein. For three sequences, the corresponding synthetic peptides were generated and attached via avidin-biotin to polylysine-condensed plasmid DNA containing a reporter gene. The addition of the peptides led to 8-14 times increase in the expression of the reporter.

Amino Acid Sequence↗
PubMed1996

Translocation of long peptides by transporters associated with antigen processing (TAP).

The major histocompatibility complex (MHC)-encoded transporters associated with antigen processing (TAP) translocate peptides from the cytosol into the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The length of class I-binding peptides is usually 8-11 amino acids, but examples of significantly longer peptides have been described. The preferred lengths and upper and lower size limits for peptides translocated by TAP have not been determined in detail because in the currently used test systems, peptides are subject to proteolytic degradation. In the present study, three sets of individual peptides or partially randomized peptide libraries ranging between 6 and 40 residues were used that contained a radiolabeled tyrosine and a consensus sequence for ER-specific N-glycosylation at opposite ends, thus ensuring that only nondegraded peptides were monitored in the transport/glycosylation assay. For three different transporters, rat TAP1/2a, rat TAP1/2u and hTAP, the most efficient ATP-dependent transport was observed for peptides with 8-12 amino acids. Hexamers and longer peptides of up to 40 amino acids were also translocated, albeit less efficiently. For two of the three sets of peptides analyzed, rat TAP1/2a showed a less stringent length selection than rat TAP1/2u and human TAP. The superior transport of the decamer of the TNKT.. Y series was not due to faster degradation or less efficient glycosylation of shorter or longer length variants. A binding assay with TAP-containing microsomes revealed a high affinity for the radiolabeled decamer (KD = 580 nM), while other length variants were clearly inferior in their binding affinities. Thus, TAP binds and preferentially translocates peptides with a length suitable for binding to MHC class I molecules, but peptides that are considerably longer may also be substrates. About 10(5) peptide binding sites per cell equivalent of microsomes were determined, providing an estimate for the number of TAP complexes in the ER membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Bifunctional recombinant proteins in cancer therapy: cell penetrating peptide aptamers as inhibitors of growth factor signaling.

The major aim of molecular cancer research is the development of new therapeutic strategies and compounds that target directly the genetic and biochemical causes of malignant transformation. Therapeutic genes, antibodies and their derivatives, but also small molecular weight compounds, have been used for this purpose. Small peptides might be able to complement these agents because of their ability to recognize specific protein domains and thus to interfere with enzymatic functions or protein-protein interactions. A variation of the yeast-two-hybrid procedure allows to select specifically binding peptides, so called peptide aptamers, from a peptide library of high complexity. This selection procedure can be adapted to any protein or protein fragment as a bait construct and selects for the intracellular interaction between the bait of choice and the peptide aptamer prey. Peptide aptamers thus selected can be cloned, provided with a protein transduction domain, expressed in bacteria and introduced into cancer cells. There they might disrupt protein-protein interactions crucial for cancer cell growth or survival. We introduce an example in which the Stat3 arm of EGF receptor signaling is selectively inhibited by a peptide aptamer and causes the growth arrest of EGF receptor-dependent tumor cells. The aptamer constructs can be supplemented with additional functional domains to enhance their inhibitory effects.

Amino Acid Sequence↗
PubMed2005

Diversity of human insulin-like growth factor (IGF) binding protein-2 fragments in plasma: primary structure, IGF-binding properties, and disulfide bonding pattern.

The insulin-like growth factor binding proteins (IGFBPs) play a major role in the regulation of the effects and the bioavailability of the insulin-like growth factors (IGFs). IGFs are released from IGFBP-IGF complexes by proteolysis of IGFBPs generating fragments with reduced ligand-binding properties. To identify naturally occurring fragments of IGFBP-2, a peptide library generated from human hemofiltrate was immunologically screened. Purification of immunoreactive IGFBP-2 fragments was performed by consecutive chromatographic steps. A total of 18 different IGFBP-2 fragments was isolated and characterized. The peptides exhibited different N-terminal amino acid residues that were located in the variable midregion of IGFBP-2. Four major cleavage sites were determined to be between Tyr103 and Gly104, Leu152 and Ala153, Arg156 and Glu157, and Gln165 and Met166. The resulting fragments were further processed by amino and/or carboxy peptidases and comprised 37-185 amino acid residues. Ligand blotting, solution binding assays, and BIAcore analyses revealed that all tested fragments retained low IGF-binding capacity. The most abundant fragment IGFBP-2 (167-279) showed 10% of IGF-II binding compared to recombinant human (rh)IGFBP-2. Furthermore, the disulfide bonding pattern of the C-terminal domain of rhIGFBP-2 was defined, indicating linkages between cysteine residues 191-225, 236-247, and 249-270. This study provides the most comprehensive molecular characterization of human IGFBP-2 fragments formed in vivo, exhibiting both residual IGF-binding capacities and the integrin-binding sequence.

Adult↗
PubMed2002

Specificity and affinity motifs for Grb2 SH2-ligand interactions.

Protein-protein interactions are often mediated by the recognition of short continuous amino acid stretches on target proteins by specific binding domains. Affinity-based selection strategies have successfully been used to define recognition motifs for a large series of such protein domains. However, in many biological systems specificity of interaction may be of equal or greater importance than affinity. To address this issue we have developed a peptide library screening technology that can be used to directly define ligands for protein domains based on both affinity and specificity of interaction. We demonstrate the value of this approach by the selection of peptide ligands that are either highly specific for the Grb2 Src homology 2 (SH2) domain or that are cross-reactive between a group of related SH2 domains. Examination of previously identified physiological ligands for the Grb2 SH2 domain suggests that for these ligands regulation of the specificity of interaction may be an important factor for in vivo ligand selection.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Solid phase combinatorial library of phosphinic peptides for discovery of matrix metalloproteinase inhibitors.

A solid phase combinatorial library of 165,000 phosphinic peptide inhibitors was prepared and screened for activity against MMP-12. The inhibitors of the library had the structure XXX-Gpsi(PO2H-CH2)L-XXX, in which X is an arbitrary amino acid and Gpsi(PO2H-CH2)L is a Gly-Leu phosphinic dipeptide analogue. The library was constructed as a one-bead-two-compounds library so that every bead contained a common quenched fluorogenic substrate and a different putative inhibitor. In addition, the inhibitor part was prepared by ladder synthesis. After incubation with MMP-12, beads containing active inhibitors were selected, and the inhibitor sequences were recorded using MALDI-TOF MS. Statistical analysis of the sequences obtained from 86 beads gave rise to a consensus sequence which was resynthesized along with 20 related sequences. Three truncated sequences and 16 sequences originally present on beads were also resynthesized. The inhibitors were investigated in an enzyme kinetic assay with MMP-12 showing that the compounds derived from the consensus sequence were strong inhibitors with Ki values down to 6 nM, whereas the sequences originally present on beads varied in potency with Ki values from micromolar to nanomolar. Truncated sequences derived from the consensus sequence were poor inhibitors of MMP-12.

Amino Acid Sequence↗
PubMed2005

Rational design of complementary peptides to the betaAmyloid 29-42 fusion peptide: an application of PepDesign.

Peptides in solution currently exist under several conformations; an equilibrium which varies with solvent polarity. Despite or because of this structure versatility, peptides can be selective biological tools: they can adapt to a target, vary conformation with solvents and so on. These capacities are crucial for cargo carriers. One promising way of using peptides in biotechnologies is to decipher their medium-sequence-structure-function relationships and one approach is molecular modelling. Only few "in silico" methods of peptide design are described in the literature. Most are used in support of experimental screening of peptide libraries. However, the way they are made does not teach us much for future researches. In this paper, we describe an "in silico" method (PepDesign) which starts by analysing the native interaction of a peptide with a target molecule in order to define which points are important. From there, a modelling protocol for the design of 'better' peptides is set. The PepDesign procedure calculates new peptides fulfilling the hypothesis, tests the conformational space of these peptides in interaction with the target by angular dynamics and goes up to the selection of the best peptide based on the analysis of complex structure properties. Experimental biological assays are finally used to test the selected peptides, hence to validate the approach. Applications of PepDesign are wide because the procedure will remain similar irrespective of the target which can be a protein, a drug or a nucleic acid. In this paper, we describe the design of peptides which binds to the fusogenic helical form of the C-terminal domain of the Abeta peptide (Abeta29-42).

Amino Acid Sequence↗
PubMed2002

Isolation and characterization of circulating fragments of the insulin-like growth factor binding protein-3.

Proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3), the major carrier of IGFs in the circulation, is an essential mechanism to regulate IGF bioavailability. To analyze naturally occurring IGFBP-3 fragments a peptide library established from human hemofiltrate was screened. Three IGFBP-3 fragments were detected with apparent molecular masses of 34, 16, and 11 kDa. Mass spectrometric and sequence analysis identified the 16 and 11 kDa peptides as glycosylated and non-glycosylated N-terminal fragments spanning residues Gly1-Ala98 of IGFBP-3. Both the circulating forms and those secreted from IGFBP-3(1-98) overexpressing cells bound IGF. Additionally, two smaller fragments (IGFBP-3(139-157) and IGFBP-3(139-159)) were identified in the hemofiltrate. The data indicate that proteolysis of circulating IGFBP-3 occurs in the variable domain at residues alanine 98, phenylalanine 138, glutamine 157, and tyrosine 159.

Amino Acid Sequence↗
PubMed2002

Phosphorylation by LAMMER protein kinases: determination of a consensus site, identification of in vitro substrates, and implications for substrate preferences.

LAMMER protein kinases are ubiquitous throughout eukaryotes, including multiple paralogues in mammals. Members are characterized by similar overall structure and highly identical amino acid sequence motifs in catalytic subdomains essential for phosphotransfer and interaction with substrates. LAMMER kinases phosphorylate and regulate the activity of the SR protein class of pre-mRNA splicing components, both in vitro and in vivo. In this study, we define an optimum in vitro consensus phosphorylation site for three family members using an oriented degenerate peptide library approach. We also examine the substrate specificity and interactions of several LAMMER protein kinases from widely diverged species with potential substrates, including their own N-termini, predicted to be substrates by the peptide-based approach. Although the optimal in vitro consensus phosphorylation site for these kinases is remarkably similar for short peptides, distinct substrate preferences are revealed by in vitro phosphorylation of intact proteins. This finding suggests that these kinases may possess varied substrates in vivo, and thus the multiple LAMMER kinases present in higher eukaryotes may perform differentiable functions. These results further demonstrate that these kinases can phosphorylate a number of substrates in addition to SR proteins, suggesting that they may regulate multiple cellular processes, in addition to the alternative splicing of pre-mRNAs.

Amino Acid Sequence↗
PubMed2003

Susceptibility of oral bacteria to an antimicrobial decapeptide.

Naturally occurring antimicrobial peptides have emerged as alternative classes of antimicrobials. In general, these antimicrobial peptides exhibit selectivity for prokaryotes and minimize the problems of engendering microbial resistance. As an alternative method to search for more effective broad-spectrum peptide antimicrobials, investigators have developed peptide libraries by using synthetic combinatorial technology. A novel decapeptide, KKVVFKVKFK (KSL), has been identified that shows a broad range of antibacterial activity. The purpose of this study was to test the efficacy of this antimicrobial peptide in killing selected strains of oral pathogens and resident saliva bacteria collected from human subjects. Cytotoxic activity of KSL against mammalian cells and the structural features of this decapeptide were also investigated, the latter by using two-dimensional NMR in aqueous and DMSO solutions. MICs of KSL for the majority of oral bacteria tested in vitro ranged from 3 to 100 microg ml(-1). Minimal bactericidal concentrations of KSL were, in general, within one to two dilutions of the MICs. KSL exhibited an ED(99) (the dose at which 99 % killing was observed after 15 min at 37 degrees C) of 6.25 microg ml(-1) against selected strains of Lactobacillus salivarius, Streptococcus mutans, Streptococcus gordonii and Actinobacillus actinomycetemcomitans. In addition, KSL damaged bacterial cell membranes and caused 1.05 log units reduction of viability counts of saliva bacteria. In vitro toxicity studies showed that KSL, at concentrations up to 1 mg ml(-1), did not induce cell death or compromise the membrane integrity of human gingival fibroblasts. NMR studies suggest that KSL adopts an alpha-helical structure in DMSO solution, which mimics the polar aprotic membrane environment, whereas it remains unstructured in aqueous medium. This study shows that KSL may be a useful antimicrobial agent for inhibiting the growth of oral bacteria that are associated with caries development and early plaque formation.

Aggregatibacter actinomycetemcomitans↗