Identification of Troponin C Antagonists from a Phage-displayed Random Peptide Library

Pierce, Heather; Schachat, Fred; Brandt, Philip W.; Lombardo, Christian R.; Kay, Brian K.

doi:10.1074/jbc.273.36.23448

Cited by 8 publications

(4 citation statements)

References 43 publications

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“…These peptides show no similarity with the peptides isolated in the present study. The affinity of our peptides is fairly typical of phage display peptides [39,40] but lower than those of peptides developed by the polysome technique [38]. However, the differences in affinity may be accounted for by differences in measuring techniques.…”

Section: Isobm-mabmentioning

confidence: 77%

Identification of novel prostate‐specific antigen‐binding peptides modulating its enzyme activity

Leinonen

Koivunen³

et al. 2000

European Journal of Biochemistry

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Prostate-specific antigen (PSA) is a serine protease with highly prostate-specific expression. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. PSA dissolves the seminal gel forming after ejaculation. It has been suggested to mediate invasion and metastasis of prostate cancer but also to exert antiangiogenic activity. We have identified peptides specific for PSA by screening cyclic phage display peptide libraries. PSA-binding peptides were isolated from four different libraries and produced as a fusion protein with glutathione S-transferase (GST). The phage and fusion proteins were shown to bind to PSA specifically as indicated by lack of binding to other serine proteinases. A peptide with four cysteines showed the highest affinity for PSA. Zn 21 , an inhibitor of PSA activity, increased the affinity of the peptides to PSA. The binding specificity was characterized by cross-inhibition using monoclonal anti-PSA antibodies of known epitope specificities. The peptides bound to the same region as mAbs specific for free PSA indicating that they bind close to the active site of the enzyme. The peptides enhanced the enzyme activity of PSA against a chromogenic substrate. These results show that peptides binding to PSA and modulating its enzyme activity can be developed by phage display technique. The peptides have the potential to be used for identification of PSA variants and for imaging and targeting of prostatic tumors.

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Section: Isobm-mabmentioning

confidence: 77%

Identification of novel prostate‐specific antigen‐binding peptides modulating its enzyme activity

Leinonen

Koivunen³

et al. 2000

European Journal of Biochemistry

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“…The second interaction site represented by the hcTnI [39–58] peptide belongs to the region 33–80 which is known to form a stable binary complex with the C‐terminal domain of cTnC [5,6]. In a completely different approach, the sequence 44–59 from hcTnI was identified by the phage‐display technology as corresponding to the minimum region of interaction [32]. However, the affinity of the biotinylated 44–59 peptide measured by BIACORE was 8.6×10 −7 M, versus 3.07×10 −9 M for the hcTnI [39–58] peptide.…”

Section: Discussionmentioning

confidence: 99%

Systematic mapping of regions of human cardiac troponin I involved in binding to cardiac troponin C: N‐ and C‐terminal low affinity contributing regions

et al. 2000

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The Spot method of multiple peptide synthesis was used to map in a systematic manner regions of the human cardiac troponin I sequence (hcTnI) involved in interactions with its physiological partner, troponin C (cTnC). Ninety-six 20-mer peptides describing the entire hcTnI sequence were chemically assembled; their reactivity with [125 I]cTnC, in the presence of 3 mM Ca 2+ , enabled the assignment of six sites of interaction (residues 19^32, 45^54, 129^138, 145^164, 161^178 and 1912 10). For several sites, a good correlation with literature data was obtained, thus validating this methodological approach. Synthetic peptides, each containing in their sequence an interaction site, were prepared. As assessed by BIACORE, all of them exhibited an affinity for cTnC in the range of 10 361 0 37 M, except for hcTnI [39^58] which showed a nanomolar affinity. This peptide was also able to block the interaction between hcTnI and cTnC. We therefore postulate that despite the existence of multiple cTnC interaction sites on the hcTnI molecule, only that region of hcTnI allows a stabilization of the complex. Residues 19^32 from the N-terminal cardio-specific extension of hcTnI were also found to be involved in interaction with cTnC; residues 19^32 may correspond to the minimal sequence of the extension which could switch between the N-and C-terminal TnC domains, depending on its phosphorylation state. Finally, two Ca 2+ -dependent cTnC binding domains within the C-terminal part of hcTnI (residues 164^178 and 191^210) were also mapped. The latter site may be linked with the cardiac dysfunction observed in stunned myocardium. ß

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“…Due to the physical linkage of the expressed peptide with its genetic sequence, libraries numbering from 10 8 to 10 10 peptides have been rapidly screened for a wide variety of applications. This useful tool has been used to map antibody epitopes and to discover peptide ligands for membrane receptors and cytosolic proteins in recent years (30). In this work, we have searched SCaM isoform (SCaM-1 and -4)-favored peptide sequences from a phage display library, and we defined novel SCaM-1-and SCaM-4-specific binding sequences.…”

Section: Cammentioning

confidence: 99%

“…First of all, three of the most frequently isolated peptides obtained from each of the SCaM-binding phages (i.e. ␣1, ␣2, and ␣3 for SCaM-1 and ␤1, ␤2, and ␤3 for SCaM-4) were expressed as GST fusion proteins using the vector pGEX-KG (30). As shown in Fig.…”

Section: Identification Of Scam-1-or -4-specific Peptides By Gelmentioning

confidence: 99%

Identification of Calmodulin Isoform-specific Binding Peptides from a Phage-displayed Random 22-mer Peptide Library

Choi

Lee

Park

et al. 2002

Journal of Biological Chemistry

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Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaMdependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca 2؉ -dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.

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Identification of Troponin C Antagonists from a Phage-displayed Random Peptide Library

Cited by 8 publications

References 43 publications

Identification of novel prostate‐specific antigen‐binding peptides modulating its enzyme activity

Identification of novel prostate‐specific antigen‐binding peptides modulating its enzyme activity

Systematic mapping of regions of human cardiac troponin I involved in binding to cardiac troponin C: N‐ and C‐terminal low affinity contributing regions

Identification of Calmodulin Isoform-specific Binding Peptides from a Phage-displayed Random 22-mer Peptide Library

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