Construction of a Multihybrid Display System: Flagellar Filaments Carrying Two Foreign Adhesive Peptides

A multivalent, bifunctional flagellum carrying two different adhesive peptides in separate flagellin subunits within a filament was constructed in Escherichia coli. The inserted peptides were the fibronectin-binding 115-mer D repeat region of Staphylococcus aureus and the 302-mer collagen-binding region of YadA of Yersinia enterocolitica. Western blotting, immunoelectron microscopy, and adhesion tests with hybrid flagella from an in trans-complemented ΔfliC E. coli strain showed that individual filaments consisted of both recombinant flagellins.     

Recognition properties of peptides hydropathically complementary to residues 356-375 of the c-raf protein

Two peptides with hydropathic complementarity to residues 356-375 of the c-raf protein were synthesized to determine if they recognize the raf-(356-375) peptide as well as the entire protein. One peptide was deduced from the complementary mRNA for the raf protein corresponding to residues 356-375, whereas the other was deduced solely from the amino acid sequence of the 20-mer segment using a computer program able to generate peptide sequences with hydropathic complementarity to a given sequence. Specific binding of both peptides to the raf 20-mer segment was demonstrated when either the raf 20-mer peptide or the complementary peptides were immobilized on a column. Binding affinities were in the millimolar-micromolar range. Identical binding properties were observed with peptides synthesized with either all D- or all L-amino acids, suggesting a lack of conformational dependence. Binding was also unaffected by the presence of 8 M urea or detergents, was dependent on solvent characteristics of pH and ionic strength, and was abolished by the presence of competing peptides in the eluting buffer. Recognition between raf complementary peptides was accompanied by spectral changes in the far and near UV region, as monitored by circular dichroism. Proteolytic degradation was retarded by the binding of these peptides. Once immobilized on a column, these peptides proved useful for the isolation by affinity chromatography of a recombinant c-raf protein from an Escherichia coli crude cell extract.     

High-throughput generation of small antibacterial peptides with improved activity

Cationic antimicrobial peptides are able to kill a broad variety of Gram-negative and Gram positive bacteria and thus are good candidates for a new generation of antibiotics to treat multidrug-resistant bacteria. Here we describe a high-throughput method to screen large numbers of peptides for improved antimicrobial activity. The method relies on peptide synthesis on a cellulose support and a Pseudomonas aeruginosa strain that constitutively expresses bacterial luciferase. A complete substitution library of 12-amino-acid peptides based on a linearized variant (RLARIVVIRVAR-NH(2)) of the bovine peptide bactenecin was screened and used to determine which substitutions at each position of the peptide chain improved activity. By combining the most favorable substitutions, we designed optimized 12-mer peptides showing broad spectrum activities with minimal inhibitory concentrations (MIC) as low as 0.5 microg/ml against Escherichia coli. Similarly, we generated an 8-mer substituted peptide that showed broad spectrum activity, with an MIC of 2 microg/ml, against E. coli and Staphylococcus aureus.     

Synthesis and antibody recognition of synthetic antigens from MUC1.

In the altered form of MUC1 mucin associated with breast cancer, the highly immunogenic sequence PDTRPAP is exposed, and may be an immunologically relevant target for the development of diagnostics or cancer immunotherapy. In this study, we report the preparation and antibody binding properties of monomeric and dimeric MUC1 peptides containing the epitope region recognized by monoclonal antibody (mAb) C595. Peptides contained a single or two copies of the whole 20-mer repeat unit (VTSAPDTRPAPGSTAPPAHG) of MUC1 protein. MUC1 40-mer peptides were prepared by the condensation of semi-protected fragments of the repeat unit, in solution or by chemical ligation. In the first case, cyclohexyl-type protecting groups were used for the synthesis of semi-protected fragments by the Boc/Bzl strategy. Unprotected fragments were used in the chemical ligation to produce thioether linkages. In one of the fragments, a Gly residue was replaced by Cys at the C-terminus and the other fragment was chloroacetylated at the N-terminus. In addition, the short peptide APDTRPAPG, and its disulfide dimer, (APDTRPAPGC)(2) were produced. The antibody binding properties of these MUC1 peptide constructs were tested by competition enzyme-linked immunosorbent assay (ELISA). The short epitope region peptide, APDTRPAPG and its dimer (APDTRPAPGC)(2) showed higher IC(50) values (IC(50) = 56.3 and 53.2 micromol/l, respectively). While the 20-mer peptide (IC(50) = 25.9 micromol/l) and more markedly its 40-mer dimers (IC(50) = 0.62 and 0.78 micromol/l) were recognized better. CD data obtained in water or in TFE indicated no significant conformational differences between the 20-mer and 40-mer peptides. We found a high level of similarity between the binding properties of the 40-mer peptides with amide or thioether links, providing a new possibility to build up oligomeric MUC1 peptides by thioether bond formation.     

Role of strong anchor residues in the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules

The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A^*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76.2%) of these peptides bound to HLA-A^*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the C-terminus were main anchor residues for HLA-A^*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A^*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer peptides to HLA-A^*2402 molecules.     

Insights on the interactions of synthetic amphipathic peptides with model membranes as revealed by 31P and 2H solid-state NMR and infrared spectroscopies

We studied the interaction between synthetic amphipathic peptides and model membranes by solid-state NMR and infrared spectroscopies. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers were synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure. To shed light on their membrane interaction, (31)P and (2)H solid-state NMR experiments were performed on both peptides in interaction with dimyristoylphosphatidylcholine vesicles in the absence and presence of cholesterol, dimyristoylphosphatidylglycerol vesicles, and oriented bicelles. (31)P NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are weakly yet markedly affected by the presence of the peptides, whereas (31)P NMR experiments on bicelles indicate no significant changes in the morphology and orientation of the bicelles. On the other hand, (2)H NMR experiments on vesicles reveal that the acyl chain order is affected differently depending on the membrane lipidic composition and on the peptide hydrophobic length. Finally, infrared spectroscopy was used to study the interfacial region of the bilayer. Based on these studies, mechanisms of membrane perturbation are proposed for the 14-mer and 21-mer peptides in interaction with model membranes depending on the bilayer composition and peptide length.     

Positioning and the specific sequence of each 13-mer motif are critical for activity of the plasmid RK2 replication origin

The minimal replication origin of the broad-host-range plasmid RK2, oriV, contains five iterons which are binding sites for the plasmid-encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT-rich region containing four 13-mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13-mer motifs have been constructed and analysed for replication activity in vivo and in vitro. The data show that the replacement of oriV 13-mers by similar but not identical 13-mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13-mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of single-nucleotide substitutions demonstrates very restricted sequence requirements depending on the 13-mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli. Our experiments demonstrate considerable complexity in the plasmid AT-rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence-specific protein interactions within the 13-mers during assembly of replication complexes at the plasmid origin.     

Interaction of nebulin SH3 domain with titin PEVK and myopalladin: implications for the signaling and assembly role of titin and nebulin

Skeletal muscle nebulin is thought to determine thin filament length and regulate actomyosin interaction in a calcium/calmodulin or S100 sensitive manner. We have investigated the binding of nebulin SH3 with proline-rich peptides derived from the 28-mer PEVK modules of titin and the Z-line protein myopalladin, using fluorescence, circular dichroism and nuclear magnetic resonance techniques. Of the six peptides studied, PR2 of titin (VPEKKAPVAPPK) and myopalladin MyoP2 (646VKEPPPVLAKPK657) bind to nebulin SH3 with micromolar affinity (approximately 31 and 3.4 microM, respectively), whereas the other four peptides bind weakly (>100 microM). Sequence analysis of titins reveals numerous SH3 binding motifs that are highly enriched in the PEVK segments of titin isoforms. Our findings suggest that titin PEVK and myopalladin may play signaling roles in targeting and orientating nebulin to the Z-line during sarcomere assembly.     

Small Retinoprotective Peptides Reveal a Receptor-binding Region on Pigment Epithelium-derived Factor

The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78–121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98–114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98–114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors.     

人类APPL2蛋白PTB结构域的表达纯化及其功能的研究

APPL蛋白,包括APPL1和APPL2,是细胞膜与细胞核之间重要的信息传递者,同时也为细胞增殖所必需。在大肠杆菌表达体系中,表达并纯化了人类APPL2蛋白PTB结构域,然后以此为靶标,利用噬菌体展示技术筛选相互作用肽段。经过3轮筛选,随机挑取48个噬菌体单克隆,进行ELISA分析。7肽序列ERLPFFY和YLTSPKH被证明能与PTB结构域特异地结合。对P3GST的野生型和将每个氨基酸突变为丙氨酸的突变型的ELISA检测,显示每个氨基酸均是结合所必需的。这些对有关APPL2的PTB结构域功能的进一步研究有参考意义。     

Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.     

Discovery of potent antimicrobial peptide analogs of Ixosin-B

Antimicrobial peptides (AMPs) represent the first defense line against infection when organisms are infected by pathogens. These peptides are generally good targets for the development of antimicrobial agents. Peptide amide analogs of Ixosin-B, an antimicrobial peptide with amino acid sequence of QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY, were designed, synthesized and examined for antimicrobial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Within the peptides synthesized, we discovered an 11-mer peptide, KRLRRVWRRWR-amide, which exhibited potent antimicrobial activity while very little hemolytic activity in human erythrocytes was observed even at high dose level (100 μM). With further modifications, this peptide could be developed into a potent antimicrobial agent in the future.     

Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome

Three tandem repeats of a 13-mer in the AT-rich region are essential to the unique replication origin of E. coli and of remotely related Enterobacteriaceae. These iterated sequences are identified by deletion analysis and sensitivities to endonucleases as the site for initial duplex opening by the initiator dnaA protein. This "open complex" requires ATP and 38 degrees C for optimum formation and stability. The subsequent dnaC-dependent entry of dnaB helicase to form a "prepriming complex" stabilizes the open structure, blocks cleavages by a restriction endonuclease in the 13-mer region, and broadens the endonuclease cutting pattern. We propose that dnaA protein recognizes and successively opens the 13-mer sequences, thereby guiding the entry of dnaB helicase into the duplex preparatory to priming of replication.     

Opposed actions of regulatory proteins, DnaA and IciA, in opening the replication origin of Escherichia coli.

The opening of the three tandem 13-mers (iterons) in the replication origin (oriC) of Escherichia coli by DnaA protein, assisted by protein HU or IHF (Hwang, D. S., and Kornberg, A. (1992) J. Biol. Chem. 267, 23083-23086), represents an essential early stage in the initiation of chromosomal replication (Bramhill, D., and Kornberg, A. (1988) Cell 54, 915-918). We now show by mutational alterations of the 13-mer region that oriC function, both in vitro and in vivo, requires AT-richness in the left 13-mer and sequence specificity in the middle and right 13-mers. Interactions of DnaA protein with the middle and right 13-mers are crucial for the opening of the region. Binding of the protein to the top strand of the 13-mers appeared to maintain single-strandedness in the bottom strand. IciA protein, the inhibitor of initiation, binds the three 13-mers and blocks the opening of the region. The degrees of inhibition by IciA protein of 13-mer opening and of oriC plasmid replication observed with mutant forms of the 13-mers could be correlated with the binding affinity of IciA protein. Whereas the binding of IciA protein to the 13-mers did not affect the binding of DnaA protein to its four 9-mers boxes, interaction of DnaA protein with the 13-mers was blocked. The selective interactions of DnaA and IciA proteins with the 13-mer region appear to be components of the on/off switch that controls initiation of E. coli chromosomal replication.     

New inhibitors of Helicobacter pylori urease holoenzyme selected from phage-displayed peptide libraries.

Urease is an important virulence factor for Helicobacter pylori and is critical for bacterial colonization of the human gastric mucosa. Specific inhibition of urease activity has been proposed as a possible strategy to fight this bacteria which infects billions of individual throughout the world and can lead to severe pathological conditions in a limited number of cases. We have selected peptides which specifically bind and inhibit H. pylori urease from libraries of random peptides displayed on filamentous phage in the context of pIII coat protein. Screening of a highly diverse 25-mer combinatorial library and two newly constructed random 6-mer peptide libraries on solid phase H. pylori urease holoenzyme allowed the identification of two peptides, 24-mer TFLPQPRCSALLRYLSEDGVIVPS and 6-mer YDFYWW that can bind and inhibit the activity of urease purified from H. pylori. These two peptides were chemically synthesized and their inhibition constants (Ki) were found to be 47 microM for the 24-mer and 30 microM for the 6-mer peptide. Both peptides specifically inhibited the activity of H. pylori urease but not that of Bacillus pasteurii.     

A novel protein binds a key origin sequence to block replication of an E. coli minichromosome

A sequence of three tandem repeats of a 13-mer in the replication origin (oriC) of E. coli is the highly conserved site of opening of the duplex for initiation of DNA synthesis. A protein that binds this sequence has been discovered in E. coli and purified to homogeneity. This novel 33 kd polypeptide behaves as a dimer. Binding to the 13-mers is specific and limited to this region. At a ratio of 10-20 monomers per oriC plasmid, the binding blocks initiation by preventing the opening of the 13-mer region by dnaA protein. Once the 13-mers are opened by dnaA protein action, the 33 kd protein is without effect on the subsequent stages of replication. The specificity of binding and profound inhibitory effect suggest a regulatory role for this protein at an early stage of chromosome initiation.     

Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies

The three-dimensional structure of the anti-apoptotic protein Bcl-xL complexed to a 25-residue peptide from the death promoting region of Bad was determined using NMR spectroscopy. Although the overall structure is similar to Bcl-xL bound to a 16-residue peptide from the Bak protein (Sattler et al., 1997), the Bad peptide forms additional interactions with Bcl-xL. However, based upon site-directed mutagenesis experiments, these additional contacts do not account for the increased affinity of the Bad 25-mer for Bcl-xL compared to the Bad 16-mer. Rather, the increased helix propensity of the Bad 25-mer is primarily responsible for its greater affinity for Bcl-xL. Based on this observation, a pair of 16-residue peptides were designed and synthesized that were predicted to have a high helix propensity while maintaining the interactions important for complexation with Bcl-xL. Both peptides showed an increase in helix propensity compared to the wild-type and exhibited an enhanced affinity for Bcl-xL.     

Binding of 8-mer to 11-mer peptides carrying the anchor residues to slow assembling HLA class I molecules (HLA-B*5101)

 The binding of 303 8-mer to 11-mer peptides carrying the anchor residues at P2 and the C-terminus to HLA-B^*5101 molecules was examined by a stabilization assay in which peptides were incubated with RMA-S-B^*5101 cells at 26 °C for 3 h. Analysis of the binding of these peptides to HLA-B^*5101 molecules showed that Pro and Ala at P2, and Ile, Val, and Leu at the C-terminus functioned as anchor residues, while Gly at P2 and Met at the C-terminus were weak anchors. Pro was a stronger anchor residue than Ala at P2, while Ile was the strongest anchor at the C-terminus. Among 8-mer to 11-mer peptides, the 9-mer peptides showed the strongest binding to HLA-B^*5101 molecules. This is in contrast to our recent findings that 10-mer and 11-mer peptides bind to HLA-B^*3501 molecules as effectively as 9-mer peptides. Since both HLA class I molecules have the same B-pocket and the binding peptides carry the same anchor residues, it is assumed that the structure of the F-pocket may restrict the length of binding peptides. The ability of HLA-B^*5101 binding peptides to stabilize the HLA-B^*5101 molecules was markedly lower than that of HLA-B^*3501 binding peptides to stabilize the HLA-B^*3501 molecules. It is known that HLA-B^*5101 is a slow assembling molecule, while HLA-B^*3501 assembles rapidly. The results imply that the slow assembling of HLA-B^*5101 molecules results from the low affinity of peptides to HLA-B^*5101 molecules. Received: 14 August 1996 / Revised: 8 October 1996     

Identification and analysis of multivalent proteolytically resistant peptides from gluten: implications for celiac sprue

Dietary gluten proteins from wheat, rye, and barley are the primary triggers for the immuno-pathogenesis of Celiac Sprue, a widespread immune disease of the small intestine. Recent molecular and structural analyses of representative gluten proteins, most notably alpha- and gamma-gliadin proteins from wheat, have improved our understanding of these pathogenic mechanisms. In particular, based on the properties of a 33-mer peptide, generated from alpha-gliadin under physiological conditions, a link between digestive resistance and inflammatory character of gluten has been proposed. Here, we report three lines of investigation in support of this hypothesis. First, biochemical and immunological analysis of deletion mutants of alpha-2 gliadin confirmed that the DQ2 restricted T cell response to the alpha-2 gliadin are directed toward the epitopes clustered within the 33-mer. Second, proteolytic analysis of a representative gamma-gliadin led to the identification of another multivalent 26-mer peptide that was also resistant to further gastric, pancreatic and intestinal brush border degradation, and was a good substrate of human transglutaminase 2 (TG2). Analogous to the 33-mer, the synthetic 26-mer peptide displayed markedly enhanced T cell antigenicity compared to monovalent control peptides. Finally, in silico analysis of the gluten proteome led to the identification of at least 60 putative peptides that share the common characteristics of the 33-mer and the 26-mer peptides. Together, these results highlight the pivotal role of physiologically generated, proteolytically stable, TG2-reactive, multivalent peptides in the immune response to dietary gluten in Celiac Sprue patients. Prolyl endopeptidase treatment was shown to abolish the antigenicity of both the 33-mer and the 26-mer peptides, and was also predicted to have comparable effects on other proline-rich putatively immunotoxic peptides identified from other polypeptides within the gluten proteome.     

PM2, a group 3 LEA protein from soybean, and its 22-mer repeating region confer salt tolerance in Escherichia coli

To have knowledge of the effect of soybean PM2 protein in protecting dehydrated cells and its functional region, PM2 cDNA was isolated from soybean immature seeds. The recombinants expressing full-length PM2, truncated polypeptides of PM2A (aa 1-262) or PM2B (aa 129-262, 22-mer repeating region), or artificial polypeptide PM2C (duplication of 22-mer repeating region) were constructed. By using SDS-PAGE and mass spectrometry approaches, these fusion polypeptides were identified and proved to be hydrophilic and heat-stable. Spot assays of BL/PM2 and BL/pET28 (as control) showed that protein PM2 increased salt tolerance (500 mM NaCl or 500 mM KCl) of Escherichia coli, rather than osmotic tolerance (1100 mM sorbitol). In addition, comparing the survival ratios of the transformants under 500 mM NaCl or 500 mM KCl stresses, the results showed that: (1) the survival ratios of BL/PM2 and BL/PM2B were quite similar, both showing much higher values than those of BL/pET28. (2) The survival ratios of BL/PM2C were much higher than those of BL/PM2, BL/PM2A, and BL/PM2B. This provides the first experimental evidence that PM2 polypeptide enhances salt tolerance of E. coli cells, and the 22-mer repeat region is an important functional region.