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At least 1,009 records · Page 56Linked to original sources
PubMed2006

[Screening of short peptides binding to cell surface interleukin-2 receptor alpha chain].

OBJECTIVE: To screen and characterize the short peptides which bind specifically to interleukin-2 (IL-2) receptor alpha chain (IL-2Ralpha) for acquisition of small antagonists for blocking the binding of IL-2 with IL-2Ralpha. METHODS: 12-mer phage displayed peptide library was screened with the target cells of MT-2 cells which expressed IL-2Ralpha at high levels. The binding phage clones were eluted by anti-IL-2Ralpha monoclonal antibody. After 3 rounds of screening, the positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, and the amino acid sequences of the positive clones were deduced from the DNA sequences. RESULTS: Seven positive clones were screened out of the 17 phage clones bound to MT-2 cells. The positive clone M15 could bind specifically to MT-2 cell and PHA-activated peripheral blood monouclear cells. Amino acid sequence analysis identified 6 sequences, all of which contained hydrophilic residues, and 5 of these 6 sequences included Tyr, Phe and Leu conservative residues. CONCLUSION: The peptide sequences containing Tyr, Phe conservative residues identified in this study can bind to cell surface IL-2Ralpha.

Amino Acid Sequence↗
PubMed2007

Activity based fingerprinting of proteases using FRET peptides.

We have successfully developed a protease assay using fluorescence resonance energy transfer based peptide libraries, which allows not only general detection of enzymatic activities, but more importantly substrate fingerprinting of proteases from different classes. The method allows the generation of substrate fingerprints of a protease from both the nonprime and prime sites. Therefore, it is well suited for profiling of major metalloproteases such as thermolysin and MMPs. We envisage that this method will provide a useful tool in the emerging field of Catalomics for high-throughput studies of proteases.

Fluorescence Resonance Energy Transfer↗
PubMed2001

Characterization of neutralizing antibodies and identification of neutralizing epitope mimics on the Clostridium botulinum neurotoxin type A.

Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 10(3) and 10(4) times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D(5)) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K(5)). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.

Amino Acid Sequence↗
PubMed1997

Using molecular repertoires to identify high-affinity peptide ligands of the WW domain of human and mouse YAP.

The WW domain is a globular protein domain that is involved in mediating protein-protein interaction and that ultimately participates in various intracellular signaling events. The domain binds to polyproline ligands containing the xPPxY consensus (where x signifies any amino acid, P is proline and Y is tyrosine). One of the first WW domain-ligand links that was characterized in vitro was the WW domain of Yes-Associated Protein (YAP) and its WBP-1 ligand. To further characterize this molecular interaction, we used two independent approaches, both of which focused on the mutational analysis of the WBP-1 ligand. We screened repertoires of synthetic decamer peptides containing the xPPxY core of WBP-1 in which all ten positions were sequentially replaced with the remaining amino acids. In addition, we screened decamer repertoires with all permutations of the amino acids which individually increased the binding to the WW domain of YAP, as compared to the wild type. In a parallel approach, we used a phage-displayed combinatorial peptide library biased for the presence of two consecutive prolines to study ligand preferences for the WW domain of YAP. Interestingly, these two lines of investigation converged and yielded the core sequence PPPPYP, which is preferred by the YAP-WW domain. This sequence was found within the p53 (tumor suppressor) binding protein-2, a probable cognate or alternative ligand interacting with YAP.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Unnatural RNA display libraries.

Combinatorial peptide and protein libraries have now been developed to accommodate unnatural amino acids in a genetically encoded format via in vitro nonsense and sense suppression. General translation features and specific regioselective and stereoselective properties of the ribosome endow these libraries with a broad chemical diversity. Alternatively, amino acid residues can be chemically derivatized post-translationally to add preferred functionality to the encoded peptide. All of these efforts are advancing combinatorial peptide and protein libraries for enhanced ligands against biological targets of interest.

Animals↗
PubMed1998

Molecular recognition properties of the C-terminal Sh3 domain of the Cbl associated protein, Cap.

A phage-displayed combinatorial peptide library was used to define the specificity of one of the three Src homology 3 (SH3) domains in a novel cytoskeletal protein, named CAP, for Cbl Associated Protein. The C-terminal SH3 domain was used to affinity select peptides with the consensus, PXPPXRXSSL, from a library of X6PXXPX6 peptides. Peptide sequences resembling this consensus were identified in two signal transduction proteins, c-Cbl and son-on-sevenless (Sos), previously shown to interact with the C-terminal SH3 domain of CAP. Genetic fusion of 16 and 14 amino acid segments of c-Cbl and Sos, respectively, to bacterial alkaline phosphatase confirmed that these segments were potential ligand sites for the C-terminal SH3 domain of CAP. Alanine-scanning mutagenesis of the c-Cbl peptide ligand confirmed that most of the residues, which were conserved among the peptide ligands selected from the combinatorial peptide library, contributed to binding to the C-terminal SH3 domain of CAP.

Amino Acid Sequence↗
PubMed2006

Convergent and divergent ligand specificity among PDZ domains of the LAP and zonula occludens (ZO) families.

We present a detailed comparative analysis of the PDZ domains of the human LAP proteins Erbin, Densin-180, and Scribble and the MAGUK ZO-1. Phage-displayed peptide libraries and in vitro affinity assays were used to define ligand binding profiles for each domain. The analysis reveals the importance of interactions with all four C-terminal residues of the ligand, which constitute a core recognition motif, and also the role of interactions with more upstream ligand residues that support and modulate the core binding interaction. In particular, the results highlight the importance of site(-1), which interacts with the penultimate residue of ligand C termini. Site(-1) was found to be monospecific in the Erbin PDZ domain (accepts tryptophan only), bispecific in the first PDZ domain of ZO-1 (accepts tryptophan or tyrosine), and promiscuous in the Scribble PDZ domains. Furthermore, it appears that the level of promiscuity within site(-1) greatly influences the range of potential biological partners and functions that can be associated with each protein. These findings show that subtle changes in binding specificity can significantly alter the range of biological partners for PDZ domains, and the insights enhance our understanding of this diverse family of peptide-binding modules.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Identification of a peptide binding motif for secreted frizzled-related protein-1.

Secreted Frizzled-related proteins (sFRPs) bind Wnts and modulate their activity. To identify putative sFRP-1 binding motifs, we screened an M13 phage displayed combinatorial peptide library. A predominant motif, L/V-VDGRW-L/V, was present in approximately 70% of the phage that bound sFRP-1. Use of peptide/alkaline phosphatase chimeras and alanine scanning confirmed that the conserved motif was important for sFRP-1 recognition. The dissociation constant for a peptide/sFRP-1 complex was 3.9 microM. Additional analysis revealed that DGR was the core of the binding motif. Although Wnt proteins lack this sequence, other proteins possessing the DGR motif may function as novel binding partners for sFRP-1.

Alanine↗
PubMed1999

Function of the transport complex TAP in cellular immune recognition.

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility complex (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. The MHC-encoded ABC transporter works in concert with the proteasome and MHC class I molecules for the antigen presentation on the cell surface for T cell recognition. TAP forms a heterodimer where each subunit consists of a hydrophilic nucleotide binding domain and a hydrophobic transmembrane domain. The transport mechanism is a multistep process composed of an ATP-independent peptide association step which induces a structural reorganization of the transport complex that may trigger the ATP-driven transport of the peptide into the endoplasmic reticulum lumen. By using combinatorial peptide libraries, the substrate selectivity and the recognition principle of TAP have been elucidated. TAP maximizes the degree of substrate diversity in combination with high substrate affinity. This ABC transporter is also unique as it is closely associated with chaperone-like proteins involved in bonding of the substrate onto MHC molecules. Most interestingly, virus-infected and malignant cells have developed strategies to escape immune surveillance by affecting TAP expression or function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Inhibition of furin by polyarginine-containing peptides: nanomolar inhibition by nona-D-arginine.

Polyarginine-containing peptides represent potent inhibitors of furin, a mammalian endoprotease that plays an important role in metabolism, activation of pathogenic toxins, and viral proliferation. The therapeutic use of D-polyarginines is especially interesting because they are not cleaved by furin and possess inhibitory potency almost equal to L-polyarginines. In this study we attempted to determine the important elements within polyarginines that contribute to effective inhibition. Structure-function analyses of polyarginine peptides showed that inhibition by polyarginine-containing peptides appeared to depend on the total number of basic charges of the positively charged inhibitors bound to the negatively charged substrate binding pocket; peptide positioning did not appear to be rigorously determined. Screening of L- and D-decapeptide positional scanning combinatorial peptide libraries indicated a preference for basic residues in nearly all positions, similar to previous results with hexapeptide libraries. Length and terminal modification studies showed that the most potent D-polyarginine tested was nona-D-arginine (D9R) amide with a K(i) of 1.3 nm. D9R amide was shown to protect RAW264.7 cells against anthrax toxemia with an IC(50) of 3.7 microm. Because of its high stability, specificity, low toxicity, small molecular weight, and extremely low K(i) against furin, D9R amide or its derivatives may represent promising compounds for therapeutic use.

Alanine↗
PubMed1998

Identification and location on syndecan-1 core protein of the epitopes of B-B2 and B-B4 monoclonal antibodies.

Using a phage display peptide library, we characterized the epitope of two monoclonal antibodies reacting with syndecan-1: B-B2 and B-B4. The identified epitopes QDIT, for B-B2, and LPEV, for B-B4, were found to align with residues 36-39 and 90-93 of the mature protein, respectively. In contrast to B-B4, the B-B2 epitope is close to a potential glycosaminoglycan attachment site. Since syndecan-1 is heavily glycosylated and post-translational modifications are cell type specific, these results might explain the differences observed in the reactivity pattern of B-B2 and B-B4 and suggest that these monoclonal antibodies are useful probes to study cell surface exposed syndecan-1.

Amino Acid Sequence↗
PubMed2000

Dissection of the ATP-binding domain of the chaperone hsc70 for interaction with the cofactor Hap46.

Several unrelated proteins are known that specifically interact with members of the mammalian hsp70 chaperone protein family independent of the hsp70 substrate-binding site. One of these is Hap46, also called BAG-1, which binds to the ATP-binding domain of hsp70 and its constitutively expressed, highly homologous counterpart hsc70, thereby affecting nucleotide binding, as well as protein folding properties, of these molecular chaperones. In an attempt to delineate the potential contact sites on hsp70/hsc70 involved in this interaction we made use of the following two independent approaches: (i) screening of membrane-bound peptide libraries based on the sequence of the ATP-binding domain and (ii) the phage-display technique with random dodecapeptides. These approaches yielded partially overlapping results and identified several possible contact regions. On the space-filling model of hsc70, the two major contact areas for Hap46 delineated in the present study are located on the same side of the molecule on either subdomain that border the central cleft harboring the nucleotide-binding site. We suggest that this bridging affects the conformation of the ATP-binding domain in a way similar to the opening of the nucleotide-binding cleft produced in the bacterial hsp70 homologue DnaK upon binding its regulatory protein GrpE.

Adenosine Triphosphate↗
PubMed2000

Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity.

The Ena-VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N-terminal EVH1 domains use groups of exposed aromatic residues to specifically recognize 'FPPPP' motifs found in the mammalian zyxin and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the affinities of these EVH1-peptide interactions are strongly dependent on the recognition of residues flanking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual K(d)s by fluorescence titration, and NMR chemical shift mapping, revealed a second affinity-determining epitope present in all four ActA EVH1-binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the affinity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1-interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.

Amino Acid Motifs↗
PubMed1998

Coloured peptides: synthesis, properties and use in preparation of peptide sub-library kits.

Several methods were developed for the solid-phase synthesis (SPPS) of coloured peptides and peptide libraries. At first a bifunctional red compound, 4-(4-(N-ethyl-N-(3-(tert-butyloxycarbonyl)aminopropyl)amino)phenylazo)be nzoic acid (Boc-EPAB), was coupled with chloromethyl resin to obtain a new solid support suitable for SPPS using Boc chemistry. Peptides synthesized on this coloured resin had the chromophore at their C-termini. N-terminally coloured peptides were synthesized on a traditional solid support, coupled with chromophoric carboxylic acid before cleavage. A model pentapeptide, Phe-Ala-Val-Leu-Gly, and its ten derivatives were synthesized and their properties studied. It was found that the presence of chromophores decreases the water solubility of peptides. However, insertion of solubilizing tags (penta-lysine sequences or polyoxyethyl chains) into the molecule of any coloured derivative resulted in enhancement of the solubility. The RP-HPLC hydrophobicity indexes (phi0) of the coloured peptides were also determined because phi0 values are closely related to their water solubility. A coloured pentapeptide library was synthesized using the portioning-mixing method. Each component of this library contained the red azo dye (EPAB) and the penta-lysine tag. Before the last coupling step the samples were not mixed. All of the 19 sub-libraries obtained after cleavage were readily soluble in water, giving intense red solutions. The effect of chromophore (EPAB) and/or penta-lysine solubilizing tag on the biological activity was also studied. Potencies of the bovine neurotensin 8-13 fragment and its different coloured and penta-lysine derivatives were compared in isolated longitudinal muscle strips of guinea pig ileum. It was shown that the hexapeptide with penta-lysine tag had almost the same activity as the 8-13 fragment itself. The activity of the EPAB-derivative was found to be rather low. However, the presence of the solubilizing tag in the coloured hexapeptide compensated the negative effect of the chromophore.

Aminobenzoates↗
PubMed2005

A novel plasma membrane Ca(2+)-pump inhibitor: caloxin 1A1.

The plasma membrane Ca(2+)-Mg(2+)-ATPase is a Ca(2+)-pump that expels Ca2+ from cells. Here we report caloxin 1A1-a novel peptide inhibitor (Ki=100 microM) of plasma membrane Ca(2+)-pump-obtained by screening a cysteine bridge-constrained random peptide library for binding to the first extracellular domain of plasma membrane Ca(2+)-pump. Dithiothreitol removed the inhibition indicating that the constraint imposed by the cysteine bridge is required for the inhibition. Caloxin 1A1 also inhibited the fast twitch sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase although weakly. Glutathione dimers (containing a cysteine bridge) inhibited the Ca(2+)-Mg(2+)-ATPase activity of sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase, but not that of plasma membrane Ca(2+)-pump. Caloxin 1A1 stabilised Ca(2+)-dependent formation of the acid stable 140-kDa acylphosphate which is a partial reaction of this enzyme. Thus caloxin 1A1 inhibits the plasma membrane Ca(2+)-pump by perturbing the first extracellular domain indicating that the transmembrane domains 1 and 2 play a role in its reaction cycle. This finding is consistent with rearrangements that occur in transmembrane helices 1 and 2 during reaction cycle of sarcoplasmic reticulum Ca(2+)-pump. Caloxin 1A1 caused an increase in cytosolic Ca2+ concentration in endothelial cells.

Amino Acid Sequence↗
PubMedDate not supplied

Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity.

To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S3 subsite and hydrophobic residues in S2; the most prevalent residue not being Phe but Val. The S1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S1'-S4' subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the kcat/KM's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO2)PMPPLCTSMK(Abz) (kcat/KM = 2109 (mM s)-1), Y(NO2)PYAVQSPQK(Abz) (kcat/KM = 1524 (mM s)-1), and Y(NO2)PVLRQQRSK(Abz) (kcat/KM = 1450 (mM s)-1). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft.

Amino Acid Sequence↗
PubMed1995

Approach to discovering novel therapeutic agents for osteoporosis based on integrin receptor blockade.

On a global scale, osteoporosis is a major and growing public health problem. In the United States, osteoporosis is present in 24 million people (mostly women) and contributes to more than 1.3 million fractures/year. Serious morbidity and mortality result from these fractures. Current therapies for osteoporosis are few, efficacy is limited, and side effects problematic. Fundamental to the pathophysiology of osteoporosis is an imbalance between the tightly coupled processes of bone resorption and bone formation that characterize normal bone remodeling. Our laboratory is engaged in a research effort focused on elucidating the role of the osteoclast integrin in bone resorption, defining the nature of ligand-integrin interactions, and developing antagonists for cell surface adhesion molecules, particularly the alpha v beta 3 vitronectin-like integrin receptor present on the surface of human osteoclasts. Peptides containing the internal arginine-glycine-aspartic acid (RGD) motif have been shown to inhibit osteoclast-mediated bone resorption in vivo. We are now designing more potent and selective inhibitors of bone resorption as a potential new mechanism-based therapeutic approach to osteoporosis based on a novel mechanism. In an effort to rapidly identify the highest affinity ligands for the human alpha v beta 3 integrin, we have generated combinatorial peptide libraries containing substantial structural diversity. For instance, based on all possible sequence combinations of extracellular matrix proteins known to bind alpha v beta 3, we recently synthesized and chemically analyzed a library of 360,000 peptides, all of which contain RGD.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Selection of D-amino-acid peptides that bind to Alzheimer's disease amyloid peptide abeta1-42 by mirror image phage display.

A mirror image phage display approach was used to identify novel and highly specific ligands for Alzheimer's disease amyloid peptide Abeta(1-42). A randomized 12-mer peptide library presented on M13 phages was screened for peptides with binding affinity for the mirror image of Abeta(1-42). After four rounds of selection and amplification the peptides were enriched with a dominating consensus sequence. The mirror image of the most representative peptide (D-pep) was shown to bind Abeta(1-42) with a dissociation constant in the submicromolar range. Furthermore, in brain tissue sections derived from patients that suffered from Alzheimer's disease, amyloid plaques and leptomeningeal vessels containing Abeta amyloid were stained specifically with a fluorescence-labeled derivative of D-pep. Fibrillar deposits derived from other amyloidosis were not labeled by D-pep. Possible applications of this novel and highly specific Abeta ligand in diagnosis and therapy of Alzheimer's disease are discussed.

Alzheimer Disease↗