Utilization of oriented peptide libraries to identify substrate motifs selected by ATM

The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine protein kinase that plays a critical role in genomic surveillance and development. Here, we use a peptide library approach to define the in vitro substrate specificity of ATM kinase activity. The peptide library analysis identified an optimal sequence with a central core motif of LSQE that is preferentially phosphorylated by ATM. The contributions of the amino acids surrounding serine in the LSQE motif were assessed by utilizing specific peptide libraries or individual peptide substrates. All amino acids comprising the LSQE sequence were critical for maximum peptide substrate suitability for ATM. The DNA-dependent protein kinase (DNA-PK), a Ser/Thr kinase related to ATM and important in DNA repair, was compared with ATM in terms of peptide substrate selectivity. DNA-PK was found to be unique in its preference of neighboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinctions between ATM and DNA-PK kinase activity. Data base searches using the library-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstream in ATM-directed signaling pathways.     

Expression,activation, and biochemical properties of a novel Arabidopsis protein kinase

An Arabidopsis SOS2 (salt overly sensitive 2)-like protein kinase gene, PKS6, was expressed in leaves, stems, and siliques, but not detectable in roots of adult plants; its expression in young seedlings was up-regulated by abscisic acid. To determine the biochemical properties of the PKS6 protein, we expressed the PKS6 coding sequence as a glutathione S-transferase fusion protein in Escherichia coli. The bacterially expressed glutathione S-transferase-PKS6 fusion protein was inactive in substrate phosphorylation. We have constructed constitutively active forms of PKS6 by either a deletion of its putative auto-inhibitory FISL motif (i.e. PKS6deltaF) or a substitution of threonine-178 with aspartic acid within the putative activation loop. We found that PKS6deltaF exhibited a strong preference for Mn2+ over Mg2+ as a divalent cation cofactor for kinase activity. PKS6DeltaF displayed substrate specificity against three different peptide substrates and had an optimal pH of approximately 7.5 and temperature optimum of 30 degrees C. The apparent Km values for ATP and the preferred peptide substrate p3 of PKS6deltaF were determined to be 1.7 and 28.5 microM, respectively. These results provide significant insights into the regulation and biochemical properties of the protein kinase PKS6. In addition, the constitutively active, gain-of-function kinase mutants will be invaluable for future determination of the in planta function of PKS6.     

Identification of a novel peptide ligand of human vascular endothelia growth factor receptor 3 for targeted tumour diagnosis and therapy

Human vascular endothelia growth factor receptor 3 (VEGFR-3) is up-regulated in a variety of human cancers. It is a potentially rational target for drug delivery. To identify novel ligands with specific binding capabilities to VEGFR-3, we screened a phage display peptide library and found a consensus motif of the peptides which is displayed by the positive phages CSDxxHxWC (x is any amino acid). The phage displaying peptide CSDSWHYWC (designated as P1) exhibited the highest affinity to VEGFR-3 in phage ELISA and the chemically synthesized P1 could bind to VEGFR-3 specifically in a dose-dependent manner. In addition, the flow cytometry assay and immunofluorescence showed that the FITC labelled P1 could bind to VEGFR-3 positive carcinoma cells with specificity. Our study suggests that P1 may be a homing peptide for treatment of tumours.     

A screening of phage displayed peptides for the recognition of fullerene (C60)

A novel approach to develop a peptide, that can recognize fullerene (C60) is described for affinity selection of phage displayed peptides from a combinatorial peptide library. Biopanning was performed using cyclic 7-mer peptide library against C60 films deposited on silicon (Si) substrate, and eluted phages with organic solvent. The phage, that recognized C60 films deposited on Si substrate, were obtained from biopanning. The nucleotides of the phage, coding a cyclic 7-mer peptide, were sequenced by standard method. Seventeen kinds of peptide displayed phages were obtained. One kind of peptide (peptide No. 4) displayed phage recognized the C60 films deposited on Si substrate. Peptide No. 4 displayed no affinity towards the Si substrate. The recognition event was monitored by a fluorescent immunoassay. Additionally, peptide No. 4 phage could recognize C60 in powder form, but not the graphite powder. This recognition event in powder form was also observed by a fluorescent immunoassay.     

小鼠cAMP依赖的蛋白激酶催化亚基α的重组表达研究

将小鼠ｃＡＭＰ依赖的蛋白激酶（ｃＡＰＫ）催化亚基α（ｍＣα）分别以成熟、麦芽糖结合蛋白（ＭＢＰ）融合以及Ｎ端连续六个组氨酸（Ｈｉｓ_６）融合的形式在大肠杆菌中得到了高效表达,且成熟及融合的重组ｍＣα均有明显的蛋白激酶活性,表明蛋白激酶催化核心结构具有相对的独立性。其中Ｈｉｓ_６－ｍＣα可利用金属离子（Ｎｉ￣（２＋））配体亲和层析（Ⅰ－ＭＡＣ）一步纯化,所得融合蛋白可通过Ｈｉｓ_６亲合手臂（Ｔａｇ）固相化于金属离子（Ｎｉ￣（２＋））配体亲和树脂上,为进一步利用ＰｈａｇｅＤｉｓｐｌａｙ多肽库筛选ｃＡＰＫ识别的底物序列和专一性抑制剂打下了基础。     

In vivo phosphorylation of a recombinant peptide substrate of CDPK suggests involvement of CDPK in plant stress responses

A phage display library displaying random peptides 15 amino acids in length was screened for peptides that interact with soybean (Glycine max L.) CDPKalpha, an isoform of calcium-dependent protein kinase (EC 2.7.1.37). Interaction of phage displaying the peptide RHPTLTRSPTLRNIQ with CDPKalpha was confirmed in an independent binding assay. A synthetic peptide corresponding to this sequence plus the surrounding amino acids AERHPTLTRSPTLRNIQPPC was synthesized and found to be a substrate of CDPK isoforms alpha, beta, and gamma. A second random peptide phage display library was constructed that displayed the substrate peptide sequence plus an additional 10 random amino acids on its amino-terminal side. Nine new peptides were obtained from the screening, all of which were phosphorylated by CDPKalpha. Sequence VSPRSFWTTWRHPTLTRSPTLRNIQ appeared twice in the screen. Because it agreed well with the consensus phosphorylation site of CDPKs, its coding sequence was cloned and stably transformed into tobacco cells. The substrate peptide expressed in tobacco was phosphorylated by recombinant CDPKalpha in vitro and by endogenous CDPK in vivo. Increased phosphorylation of the peptide substrate in response to hydrogen peroxide treatment was observed in transgenic tobacco cells. These results show that the peptide substrate expressed in tobacco cells can be used as a CDPK activity reporter for in vivo studies.     

A novel peptide, THALWHT, for the targeting of human airway epithelia

Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on airway epithelial cells, a phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr-His-Ala-Leu-Trp-His-Thr (THALWHT). Subsequent binding studies confirmed that THALWHT-displaying phages bound much stronger than phages displaying control peptides to human airway epithelial cells. In contrast, no significant binding differences were observed on a variety of non-airway-derived human cell lines suggesting selective binding of the THALWHT motif to airway epithelia. Confocal microscopy of such cells after exposure to labelled synthetic THALWHT peptide indicated that its binding is followed by specific internalisation via endocytosis. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA binding moiety enabled efficient targeted gene delivery into human airway epithelial cells. Competition assays with free THALWHT peptide confirmed the specificity of gene delivery. Thus, the THALWHT motif may prove a useful targeting moiety for both non-viral and viral gene therapy vectors.     

Identification of epitopes of trichosanthin by phage peptide library

The phage displayed random peptide library has recently emerged as a powerful technique for analyzing Ab-Ag interactions. In this study, the method was employed to identify epitopes of trichosanthin. Two monoclonal Abs (4B5, 2E9) which recognized different epitopes of trichosanthin (TCS) were selected and a phage-peptide library with nine amino acids (9 aa) was used to screen the positive phage clones that have high affinity to the mAbs. Two groups of phage clones that carried peptide-specific binding to mAbs were identified by the screen. The identified phage clones carried peptide-specific binding to 4B5 and 2E9 mAbs were immunized in mice. To evaluate mimotope of selected phages, the specific binding activity to TCS was measured in the serum from phage-immunized mice. They all showed positive results. The conserved interaction motifs were deduced from the peptide sequences of each group of selected phage clones. When compared the motif sequence with the sequence of TCS, it was predicted that 4B5-corresponding epitope was located at 27-37 aa of TCS protein and 2E9-corresponding epitope was located at 41-48 aa of TCS. The predicted sequence of 4B5-corresponding epitope was further confirmed by site-directed mutation of TCS protein. The data showed that the expressed TCS protein mutated in 4B5-corresponding epitope was unable to bind 4B5 mAb. The results suggested that the phage display peptide library is useful to identify Ag epitopes and to raise Ab in disease diagnosis and treatment.     

Construction and characterization of a 9-mer phage display pVIII-library with regulated peptide density

The construction of a new phagemid vector for display of peptides on the pVIII major coat protein of filamentous bacteriophage is described, in which expression of pVIII-peptide fusions was placed under the control of the arabinose-inducible P_BAD promoter. The new phagemid showed excellent capacity for the regulation of peptide expression, as judged by enzyme-linked immunosorbent assay (ELISA) and electron microscopy of immunogold-labeled FLAG peptides displayed on phages. Regulation of the density of peptide fusions displayed on phages may offer advantages in the search for new peptide ligands due to the possibility of regulating the stringency of binding, reducing selection based on avidity effects during biopanning. Furthermore, the peptide expression in the absence of inducer was effectively shut off, minimizing growth bias of individual clones. A 9-mer phage display library prepared using the constructed phagemid was generated by insertion of randomly synthesized oligonucleotides close to the N-terminal of the pVIII protein. The library comprised a total of 9.4 × 10⁹ unique transformants, and was confirmed to show high diversity. The functional utility of the library was confirmed by the successful affinity selection of peptides binding to matrix metalloproteinase-9 (MMP-9). The majority of selected peptides shared the consensus motif R(D/N)XXG(M/L)(V/I)XQ, not previously selected during biopanning against MMP-9.     

Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.     

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.     

Purification and characterization of a novel proline-directed protein kinase from bovine brain.

A novel protein kinase which phosphorylates a synthetic peptide substrate (RRPDAHRTPNRAF) has been purified approximately 200,000-fold from bovine brain. This peptide contains the consensus sequence for phosphorylation by the p34cdc2 kinase. The purification procedure took advantage of the phenomenon that this novel brain kinase, in partially purified extracts, chromatographed on a gel filtration column as a high molecular weight complex which dissociated in buffer containing 1 M NaCl. The purified native enzyme was estimated to be approximately 63,000, and displayed two bands of M(r) = 33,000 and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On Western immunoblot, the M(r) = 33,000 peptide reacted strongly with antibodies specific for a conserved amino-terminal sequence, weakly with antibodies to the conserved PSTAIRE sequence, and not at all with antibodies to the carboxyl terminus, of HeLa cell p34cdc2. The brain kinase and p34cdc2 were similar in displaying good activity toward the parent peptide substrate, but no activity toward peptide analogues in which the -T-P- motif was substituted with either -T-G- or -T-A-. Both kinases showed marked preference in phosphorylating a peptide derived from H1 histone (KTPKKAKKPKTPKKAKKL), and both kinases could be phosphorylated by the src-family tyrosine kinase, p56lyn, purified from bovine spleen. However, the brain kinase did not co-purify with a subunit having a molecular weight corresponding to known cyclins, nor did it undergo specific interaction with p13suc1 beads, suggesting that this enzyme is distinct from p34cdc2.     

Potent inhibitory ligands of the GRB2 SH2 domain from recombinant peptide libraries.

We cloned and expressed the SH2 domain of human GRB2 as glutathione S-transferase and maltose binding protein fusion proteins. We screened three phagemid-based fd pVIII-protein phage display libraries against SH2 domain fusion proteins. Sequence analysis of the peptide extensions yielded a variety of related peptides. By examining the ability of the phage clones to bind other SH2 domains, we demonstrated that the phage were specific for the SH2 domain of GRB2. Based on the sequence motif identified in the "random" library screening experiment, we also built and screened a phage display library based on a Tyr-X-Asn motif (X5-Tyr-X-Asn-X8). To examine the affinity of the phage derived peptides for GRB2, we set up a radioligand competition binding assay based on immobilized GRB2 and radiolabelled autophosphorylated EGFR ICD as the radioligand. Results obtained with peptide competitors derived from the phage sequences demonstrated that nonphosphotyrosine-containing peptides identified with the phage display technology had an affinity for the receptor similar to tyrosine-phosphorylated peptides derived from the EGFR natural substrate. Interestingly, when the phage display peptides were then phosphorylated on tyrosine, their affinity for GRB2 increased dramatically. We also demonstrated the ability of the peptides to block the binding of the GRB2 SH2 domain to EGFR in a mammalian cell-based binding assay.     

Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4.

Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead. Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes. Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies. The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenicoutercapsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4. Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase interaction sites. In further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro.The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues). As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site. Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly internalizes terminase regions within the portal in the packasome complex. Both similarities and differences are seen in comparison to analogous sites which have been identified in phages T3 and lambda.     

Structure and phosphorylation of eukaryotic initiation factor 2. Casein kinase 2 and protein kinase C phosphorylate distinct but adjacent sites in the beta-subunit

Eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes can be phosphorylated on its beta-subunit by two different protein kinases, protein kinase C and casein kinase 2. Phosphorylation by these kinases is additive, suggesting that they phosphorylate different sites (serine residues) in eIF-2 beta. Two-dimensional peptide mapping of the phosphopeptides generated from labelled eIF-2 beta by digestion with trypsin, cyanogen bromide or Staphylococcus aureus V8 proteinase showed that protein kinase C and casein kinase 2 phosphorylated distinct and different sites in this protein. This conclusion was supported by the results of analysis of the phosphopeptides on reverse-phase chromatography. Analysis of the phosphopeptides derived from eIF-2 beta labelled by both kinases together strongly suggested that the sites labelled by protein kinase C and casein kinase 2 are adjacent in the primary sequence. These data are discussed in the light of the present understanding of the sequence specificity of the kinases. Rat liver eIF-2 beta was also found to be a substrate for protein kinase C and casein kinase 2, which were again shown to label different serine residues.     

Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation

The hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase required for replication of the HCV RNA genome. We have identified a peptide that most closely resembles a short region of the protein kinase C-related kinase 2 (PRK2) by screening of a random 12-mer peptide library displayed on the surface of the M13 bacteriophage with NS5B proteins immobilized on microwell plates. Competitive phage enzyme-linked immunosorbent assay with a synthetic peptide showed that the phage clone displaying this peptide could bind HCV RNA polymerase with a high affinity. Coimmunoprecipitation and colocalization studies demonstrated in vivo interaction of NS5B with PRK2. In vitro kinase assays demonstrated that PRK2 specifically phosphorylates NS5B by interaction with the N-terminal finger domain of NS5B (amino acids 1-187). Consistent with the in vitro NS5B-phosphorylating activity of PRK2, we detected the phosphorylated form of NS5B by metabolic cell labeling. Furthermore, HCV NS5B immunoprecipitated from HCV subgenomic replicon cells was specifically recognized by an antiphosphoserine antibody. Knock-down of the endogenous PRK2 expression using a PRK2-specific small interfering RNA inhibited HCV RNA replication. In contrast, PRK2 overexpression, which was accompanied by an increase of in the level of its active form, dramatically enhanced HCV RNA replication. Altogether, our results indicate that HCV RNA replication is regulated by NS5B phosphorylation by PRK2.