Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display

The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) downward arrow (L/Y)(T/V/M/R)(R/K), where downward arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E downward arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest kcat/Km values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower kcat/Km values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer.     

Identification of a novel calpain inhibitor using phage display

Calpains are calcium- and thiol-dependent proteases that cleave a variety of intracellular substrates. Overactivation of the calpains has been implicated in a number of diseases and conditions such as ischemic stroke indicating a need for the development of calpain inhibitors. A major problem with current calpain inhibitors has been specific targeting to calpain. To identify highly specific calpain interacting peptides, we developed a peptide-phage library screening method based on the calcium-dependent conformation change associated with calpain activation. A phage-peptide library representing greater than 2 billion expressed 12-mers was incubated with calpain I in the presence of calcium. The calcium-dependent bound phage was then eluted by addition of EGTA. After four rounds of selection we found a conserved 5-mer sequence represented by LSEAL. Synthetic LSEAL inhibited tau-calpain interaction and in vitro proteolysis of tau- and alpha-synuclein by calpains. Deletion of the portion of the tau protein containing a homologous sequence to LSEAL resulted in decreased calpain-mediated tau degradation. These data suggest that these peptides may represent novel calpastatin mimetics.     

Identification of peptide inhibitors of penicillinase using a phage display library

There is a constant need to identify novel inhibitors to combat β-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM⁻¹) and binding affinity (K_D = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV–vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a K_i of 9.22 μM and a K_i′ of 33.12 μM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of β-lactamases and development of novel inhibitors of β-lactamases.     

Determination of the substrate specificity of tripeptidyl-peptidase I using combinatorial peptide libraries and development of improved fluorogenic substrates

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1' and P2' residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.     

Identification of peptide sequences that target to the brain using in vivo phage display

Phage display technology could provide a rapid means for the discovery of novel peptides. To find peptide ligands specific for the brain vascular receptors, we performed a modified phage display method. Phages were recovered from mice brain parenchyma after administrated with a random 7-mer peptide library intravenously. A longer circulation time was arranged according to the biodistributive brain/blood ratios of phage particles. Following sequential rounds of isolation, a number of phages were sequenced and a peptide sequence (CTSTSAPYC, denoted as PepC7) was identified. Clone 7-1, which encodes PepC7, exhibited translocation efficiency about 41-fold higher than the random library phage. Immunofluorescence analysis revealed that Clone 7-1 had a significant superiority on transport efficiency into the brain compared with native M13 phage. Clone 7-1 was inhibited from homing to the brain in a dose-dependent fashion when cyclic peptides of the same sequence were present in a competition assay. Interestingly, the linear peptide (ATSTSAPYA, Pep7) and a scrambled control peptide PepSC7 (CSPATSYTC) did not compete with the phage at the same tested concentration (0.2-200?pg). Labeled by Cy5.5, PepC7 exhibited significant brain-targeting capability in in vivo optical imaging analysis. The cyclic conformation of PepC7 formed by disulfide bond, and the correct structure itself play a critical role in maintaining the selectivity and affinity for the brain. In conclusion, PepC7 is a promising brain-target motif never been reported before and it could be applied to targeted drug delivery into the brain.     

Identification of ligand-selective peptide antagonists of the mineralocorticoid receptor using phage display

The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily. Pathological activation of the MR causes cardiac fibrosis and heart failure, but clinical use of MR antagonists is limited by the renal side effect of hyperkalemia. The glucocorticoid cortisol binds the MR with equivalent affinity to that of the mineralocorticoids aldosterone and deoxycorticosterone. In nonepithelial tissues, including the myocardium, which do not express the cortisol-inactivating enzyme 11β hydroxysteroid dehydrogenase 2, cortisol has been implicated in the activation of MR. The mechanisms for ligand- and tissue-specific actions of the MR are undefined. Over the past decade, it has become clear that coregulator proteins are critical for nuclear receptor-mediated gene expression. A subset of these coregulators may confer specificity to MR-mediated responses. To evaluate whether different physiological ligands can induce distinct MR conformations that underlie differential coregulator recruitment and ligand-specific gene regulation, we utilized phage display technology to screen 10(8) 19mer peptides for their interaction with the MR in the presence of agonist ligands. We identified ligand-selective MR-interacting peptides that acted as potent antagonists of MR-mediated transactivation. This represents a novel mechanism of MR antagonism that may be manipulated in the rational design of a ligand- or tissue-selective MR modulator to treat diseases like heart failure without side effects such as hyperkalemia.     

The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage

Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) protein family that was recently identified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes purified from interleukin-1-stimulated bovine nasal cartilage (Tortorella, M. D., Burn, T. C., Pratta, M. A. , Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., and Arner, E.C. (1999) Science 284, 1664-1666; 2 Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., Wynn, R., Duke, J. L., George, H. J., Hillman, M. C., Jr., Murphy, K., Wiswall, B. H., Copeland, R. A., Decicco, C. P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Burn, T. C. (1999) J. Biol. Chem. 274, 23443-23450). The aggrecan products generated by this enzyme are found in cartilage cultures stimulated with cytokines and in synovial fluid from patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the thrombospondin type-1 (TSP-1) motif located within the C terminus of aggrecanase-1 binds to the glycosaminoglycans of aggrecan. Data from several studies indicate that this binding of aggrecanase-1 to aggrecan through the TSP-1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated form of aggrecanase-1 lacking the TSP-1 motif was not effective in cleaving aggrecan. 2) Several peptides representing different regions of the TSP-1 motif effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective in cleaving glycosaminoglycan-free aggrecan. Taken together, these data suggest that the TSP-1 motif of aggrecanase-1 is critical for substrate recognition and cleavage.     

Identification of a methotrexate-binding peptide from a T7 phage display screen using a QCM device

An 11-mer unique peptide sequence SIFPLCNSGAL was identified as a methotrexate (MTX)-binding peptide from a T7 phage display screen using a quartz-crystal microbalance (QCM) biosensor. The synthetic peptide displayed weak interaction with MTX (K(D) 2.23x10(-5) M) using surface plasmon resonance (SPR). Interestingly, analysis of the primary amino acid sequence of the peptide identified similarities to the MTX-binding site of dihydrofolate reductase (DHFR). Our results highlight the importance of this primary sequence for the recognition of the MTX molecule.     

Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library

4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca(2+) -dependent chaperone and a Ca(2+) -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K(d) = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein.     

Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide

A fluorogenic substrate for vertebrate collagenase and gelatinase, Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2, was designed using structure-activity data obtained from studies with synthetic inhibitors and other peptide substrates of collagenase. Tryptophan fluorescence was efficiently quenched by the NH2-terminal dinitrophenyl group, presumably through resonance energy transfer. Increased fluorescence accompanied hydrolysis of the peptide by collagenase or gelatinase purified from culture medium of porcine synovial membranes or alkali-treated rabbit corneas. Amino acid analysis of the two product peptides showed that collagenase and gelatinase cleaved at the Gly-Leu bond. The peptide was an efficient substrate for both enzymes, with kcat/Km values of 5.4 microM-1 h-1 and 440 microM-1 h-1 (37 degrees C, pH 7.7) for collagenase and gelatinase, respectively. Under the same conditions, collagenase gave kcat/Km of about 46 microM-1 h-1 for type I collagen from calf skin. Since both enzymes exhibited similar Km values for the synthetic substrate (3 and 7 microM, respectively), the higher catalytic efficiency of gelatinase reflects predominantly an increase in kcat. Both enzymes were inhibited by HSCH2(R,S)CH[CH2CH(CH3)2]CO-L-Phe-L-Ala-NH2 in this assay (50% inhibition at 20 nM and less than 1 nM for collagenase and gelatinase, respectively). Soluble type I collagen was a competitive inhibitor of peptide hydrolysis by collagenase (KI = 0.8 microM) and exhibited mixed inhibition of gelatinase (KI = 0.3 microM).     

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.     

Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.     

Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin

We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmin's physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.     

A novel PCNA-binding motif identified by the panning of a random peptide display library

Proliferating cell nuclear antigen (PCNA) has recently been identified as a target for the binding of proteins involved in DNA replication, DNA repair, and cell cycle control. The interactions between PCNA and a number of these proteins are known to be mediated by a conserved peptide motif. In this study, a random peptide library in which peptide sequences are displayed on the E. coli bacterial flagellin protein was screened for PCNA-binding sequences. Analysis of the retrieved peptide sequences verified the presence of the known PCNA-binding motif. In addition, a second, larger group of peptides containing a different consensus sequence for PCNA binding was discovered. This sequence was found to be present on DNA polymerase delta, and a peptide conforming to this sequence was demonstrated to bind to PCNA. Database search and analysis show that many proteins contain the second consensus sequence. These include proteins that are involved in DNA replication, repair, and cell cycle control. The demonstration of this second PCNA-binding motif may provide a basis for identifying and experimentally testing specific proteins for the structural basis for PCNA binding.     

Screening and identification of a targeting peptide to hepatocarcinoma from a phage display peptide library

Ligands specific to cell surface receptors have been heavily investigated in cancer research. Phage display technology is a powerful tool in this field and may impact clinical issues including functional diagnosis and targeted drug delivery. In this study, a hepatocellular carcinoma cell line (HepG2) and a normal hepatocyte line (L-02) were used to carry out subtractive screening in vitro with a phage display-7 peptide library. After four rounds of panning, there was an obvious enrichment for the phages specifically binding to the HepG2 cells, and the output/input ratio of phages increased about 976-fold (from 0.3x10(-7) to 292.8x10(-7)). A group of peptides capable of binding specifically to the hepatoma cells were obtained, and the affinity of these peptides to the targeting cells and tissues was studied. Through a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and immunofluorescence, the S1 phage and synthetic peptide HCBP1 (sequence FQHPSFI) were shown to bind to the tumor cell surfaces of two hepatoma cell lines and biopsy specimens, but not to normal hepatocytes, other different cancer cells, or nontumor liver tissues. In conclusion, the peptide HCBP1 may be a potential candidate for targeted drug delivery in therapy of hepatoma cancer.     

Identification of ChChd3 as a novel substrate of the cAMP-dependent protein kinase (PKA) using an analog-sensitive catalytic subunit

Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N(6)-substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr(197) and Ser(338). Based on its kinetic properties, the engineered C-subunit preferred N(6)(benzyl)-ATP and N(6)(phenethyl)-ATP over other ATP analogs, but still retained a 30 microm K(m) for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins.     

Kinetic analysis of the cleavage of human protease-activated receptor-1 / 2 / 3 and 4 using quenched-fluorescent peptide substrates

Protease-activated receptors [PARs] are a family of G-protein-coupled seven-transmembrane domain receptors that are activated by proteolytic cleavage of their amino-terminal exodomain. To characterize the cleavage rate of human PAR-1 / 2 / 3 and 4 by trypsin and thrombin, four synthetic quenched-fluorescent peptide substrates have been synthesized. Each substrate consisted of a ten-residue peptide spanning the receptor activation cleavage site and using progress-curve kinetics, k(cat) / K(m) values were determined.     

Caspase cleavage of initiation factor 4E-binding protein 1 yields a dominant inhibitor of cap-dependent translation and reveals a novel regulatory motif

Eukaryotic initiation factor 4E (eIF4E) binding proteins (4E-BPs) regulate the assembly of initiation complexes required for cap-dependent mRNA translation. 4E-BP1 undergoes insulin-stimulated phosphorylation, resulting in its release from eIF4E, allowing initiation complex assembly. 4E-BP1 undergoes caspase-dependent cleavage in cells undergoing apoptosis. Here we show that cleavage occurs after Asp24, giving rise to the N-terminally truncated polypeptide Delta4E-BP1, which possesses the eIF4E-binding site and all the known phosphorylation sites. Delta4E-BP1 binds to eIF4E and fails to become sufficiently phosphorylated upon insulin stimulation to bring about its release from eIF4E. Therefore, Delta4E-BP1 acts as a potent inhibitor of cap-dependent translation. Using a mutagenesis approach, we identify a novel regulatory motif of four amino acids (RAIP) which lies within the first 24 residues of 4E-BP1 and which is necessary for efficient phosphorylation of 4E-BP1. This motif is conserved among sequences of 4E-BP1 and 4E-BP2 but is absent from 4E-BP3. Insulin increased the phosphorylation of 4E-BP3 but not sufficiently to cause its release from eIF4E. However, a chimeric protein that was generated by replacing the N terminus of 4E-BP3 with the N-terminal sequence of 4E-BP1 (containing this RAIP motif) underwent a higher degree of phosphorylation and was released from eIF4E. This suggests that the N-terminal sequence of 4E-BP1 is required for optimal regulation of 4E-BPs by insulin.     

Identification of a RON tyrosine kinase receptor binding peptide using phage display technique and computational modeling of its binding mode

RON (Recepteur d’Origine Nantais) tyrosine kinase receptor is a promising target for therapeutic intervention in cancer therapy. The aim of this work was identification of RON-binding peptides using phage display and computational modeling their mode of binding. A 12-mer peptide phage library was utilized to perform biopanning against RON. The RON-binding ability of the selected peptide-displaying phage and their possible binding sites were examined by ELISA. Binding modes and affinities were also predicted by docking and molecular dynamics (MD) simulation. The results of ELISA experiment showed that P6 peptide displaying phage has higher affinity for RON compared to others and its binding site is located out of ligand binding site. Docking and MD simulation results also indicated higher affinity of P6 to RON as well as its exosite-binding feature. Taken together, our data suggest a capacity for P6 peptide (FEHSLYKEMTHL) to be utilized as RON binding agent, and hence be used for various purposes, including design of drug delivery systems for transferring cytotoxic agents to RON-positive cancer cells, interfering with RON signaling, peptidomimetics design, and diagnostic imaging.