人可溶性B淋巴细胞刺激因子结合小肽的筛选及其免疫抑制功能分析

目的筛选和鉴定B淋巴细胞刺激因子(BLyS)的抑制性小肽。方法用重组人BLyS筛选噬菌体构象型7肽库、人工合成小肽鉴定其免疫抑制功能。结果经过筛选，得到两个阳性噬菌体克隆．人工合成的两个小肽在体外能特异性地抑制BLyS刺激细胞增生的活性。结论获得了两个能够抑制BLyS功能的小肽。     

从人肝癌细胞cDNA表达文库筛选人肝再生增强因子相互作用蛋白及其活性初步鉴定

目的利用噬菌体表面展示技术筛选人肝再生增强因子(hALR)相互作用蛋白并初步鉴定噬菌体展示肽的生物学活性。方法以hALR为靶蛋白,采用T7噬菌体表面展示技术从人肝癌细胞cDNA表达文库筛选与 hALR相互作用的特异噬菌体克隆;对获得的特异噬菌体克隆cDNA插入片段进行测序及生物信息学分析;利用3H- TdR掺入法测定噬菌体展示肽及联合hALR对QGY肝癌细胞的增殖效应。结果经过四轮生物淘洗,特异噬菌体克隆富集,获得212 bp的噬菌体cDNA插入序列,生物信息学分析与人Citron激酶同源性达100％;该噬菌体展示肽及联合应用hALR对QGY细胞具有促增殖效应。结论利用噬菌体表面展示技术可以筛选出与hALR具有相互作用的多肽,该序列与人Citron激酶完全同源,Citron激酶可能参与了hALR促肝癌细胞增殖的过程。     

Systematic isolation of peptide signal molecules regulating development in hydra: LWamide and PW families

To isolate new peptide signal molecules involved in regulating developmental processes in hydra, a novel screening project was developed. Peptides extracted from the tissue of Hydra magnipapillata were systematically purified to homogeneity using HPLC. A fraction of each purified peptide was examined by differential display–PCR for its ability to affect gene expression in hydra. Another fraction was used to determine the tentative structure using an amino acid sequence analyzer and/or a mass spectrometer. Based on the results, peptides of potential interest were selected for chemical synthesis, followed by confirmation of the identity of the synthetic with the native peptides using HPLC. Using this approach, 286 peptides have been isolated, tentative amino acid sequences have been determined for 95 of them, and 19 synthetic peptides identical to native ones were produced. The 19 synthetic peptides were active in a variety of biological tests. For example, Hym-54 stimulated muscle contraction in adult polyps of hydra and sea anemone, Anthopleura fuscoviridis, and induced metamorphosis of planula, the larval stage, into polyps in a marine hydrozoan species, Hydractinia serrata. Another peptide, Hym-33H, inhibited nerve cell differentiation in hydra and induced tissue contraction in planula of Hydractinia serrata. The evidence obtained so far suggests that hydra contains a large number (>350) of peptide signal molecules involved in regulating developmental or other processes in cnidaria. These peptides can be isolated and their functions examined systematically with the new approach developed in this study.     

Rational combinatorial design of pore-forming beta-sheet peptides

Exogenous polypeptides that self-assemble on biological membranes into pores are abundant and structurally diverse, functioning as transporters, toxins, ion channels, and antibiotics. A means for designing novel pore-forming sequences would unlock new opportunities for the development and engineering of protein function in membranes. Toward this goal, we designed a 9,604-member rational combinatorial peptide library based on the structural principles of known membrane-spanning beta-sheets. When the library was screened under stringent conditions for sequences with pore-forming activity, a single active motif was found, which is characterized by aromatic residues at the lipid-exposed interfacial positions and basic residues in the pore-lining portion of the sequence. Peptides with this motif assembled on bilayer membranes into beta-sheets and formed transient peptide/lipid pores of approximately 1-nm diameter. The mechanism of action is very similar to that of natural, pore-forming peptides. These methods provide a powerful means for selecting and engineering novel pore-forming sequences and will open prospects for designing peptide antibiotics, biosensors, and new membrane protein structures.     

Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA

Several mutations that cause severe forms of the human disease autosomal dominant retinitis pigmentosa cluster in the C-terminal region of rhodopsin. Recent studies have implicated the C-terminal domain of rhodopsin in its trafficking on specialized post-Golgi membranes to the rod outer segment of the photoreceptor cell. Here we used synthetic peptides as competitive inhibitors of rhodopsin trafficking in the frog retinal cell-free system to delineate the potential regulatory sequence within the C terminus of rhodopsin and model the effects of severe retinitis pigmentosa alleles on rhodopsin sorting. The rhodopsin C-terminal sequence QVS(A)PA is highly conserved among different species. Peptides that correspond to the C terminus of bovine (amino acids 324–348) and frog (amino acids 330–354) rhodopsin inhibited post-Golgi trafficking by 50% and 60%, respectively, and arrested newly synthesized rhodopsin in the trans-Golgi network. Peptides corresponding to the cytoplasmic loops of rhodopsin and other control peptides had no effect. When three naturally occurring mutations: Q344ter (lacking the last five amino acids QVAPA), V345M, and P347S were introduced into the frog C-terminal peptide, the inhibitory activity of the peptides was no longer detectable. These observations suggest that the amino acids QVS(A)PA comprise a signal that is recognized by specific factors in the trans-Golgi network. A lack of recognition of this sequence, because of mutations in the last five amino acids causing autosomal dominant retinitis pigmentosa, most likely results in abnormal post-Golgi membrane formation and in an aberrant subcellular localization of rhodopsin.     

Identification of peptides targeting the surface of Plasmodium falciparum-infected erythrocytes using a phage display peptide library

Drastic changes in the plasma membrane of Plasmodium falciparum-infected red blood cells (iRBCs) make the surface of iRBCs distinct from that of the uninfected erythrocyte. To identify small peptides that would specifically recognize the altered surface of iRBCs, we screened a phage display peptide library (PDL) on the surface of iRBCs. After the sixth panning of the PDL, eight phage clones of 18 sequenced clones had the same sequence, LVDAAAL (named P1) and specific binding of P1 to the surface of iRBCs was confirmed using phage expressing P1 peptides and synthetic P1 peptide. When P1 peptide was conjugated with a peptide having moderate hemolytic activity, the peptide conjugate inhibited the growth of intracellular parasites in a dose-dependent manner, whereas control peptides were without effect. Our results demonstrate that the P1 peptide may be a lead compound for the development of anti-malarial agents targeting the surface of iRBCs.     

Peptide modulators of alpha‐glucosidase

Aims/Introduction

Acute glucose fluctuations during the postprandial period pose great risk for cardiovascular complications and thus represent an important therapeutic approach in type 2 diabetes. In the present study, screening of peptide libraries was used to select peptides with an affinity towards mammalian intestinal alpha-glucosidase as potential leads in antidiabetic agent development.

Materials and Methods

Three phage-displayed peptide libraries were used in independent selections with different elution strategies to isolate target-binding peptides. Selected peptides displayed on phage were tested to compete for an enzyme-binding site with known competitive inhibitors, acarbose and voglibose. The four best performing peptides were synthesized. Their binding to the mammalian alpha-glucosidase and their effect on enzyme activity were evaluated.

Results

Two linear and two cyclic heptapeptides with high affinity towards intestinal alpha-glucosidase were selected. Phage-displayed as well as synthetic peptides bind into or to the vicinity of the active site on the enzyme. Both cyclic peptides inhibited enzyme activity, whereas both linear peptides increased enzyme activity.

Conclusions

Although natural substrates of glycosidase are polysaccharides, in the present study we successfully isolated novel peptide modulators of alpha-glucosidase. Modulatory activity of selected peptides could be further optimized through peptidomimetic design. They represent promising leads for development of efficient alpha-glucosidase inhibitors.     

The molecular mechanism of lipid monolayer collapse

Lipid monolayers at an air–water interface can be compressed laterally and reach high surface density. Beyond a certain threshold, they become unstable and collapse. Lipid monolayer collapse plays an important role in the regulation of surface tension at the air–liquid interface in the lungs. Although the structures of lipid aggregates formed upon collapse can be characterized experimentally, the mechanism leading to these structures is not fully understood. We investigate the molecular mechanism of monolayer collapse using molecular dynamics simulations. Upon lateral compression, the collapse begins with buckling of the monolayer, followed by folding of the buckle into a bilayer in the water phase. Folding leads to an increase in the monolayer surface tension, which reaches the equilibrium spreading value. Immediately after their formation, the bilayer folds have a flat semielliptical shape, in agreement with theoretical predictions. The folds undergo further transformation and form either flat circular bilayers or vesicles. The transformation pathway depends on macroscopic parameters of the system: the bending modulus, the line tension at the monolayer–bilayer connection, and the line tension at the bilayer perimeter. These parameters are determined by the system composition and temperature. Coexistence of the monolayer with lipid aggregates is favorable at lower tensions of the monolayer–bilayer connection. Transformation into a vesicle reduces the energy of the fold perimeter and is facilitated for softer bilayers, e.g., those with a higher content of unsaturated lipids, or at higher temperatures.     

Structural evolution of urotensin-I: Reflections of life before corticotropin releasing factor

Peptides have a long evolutionary history that predates the appearance of metazoans. The corticotropin releasing factor (CRF) family of peptides is among the most ancient peptide lineages. The identification and characterization of urotensin-I and related orthologues led the way for the elucidation of the entire CRF peptide family. A comparative analysis of the CRF paralogue sequences suggest that CRF is the most derived of these peptides and has lost many of its ancestral characteristics after it became associated with the hypothalamic-pituitary-adrenal/interrenal (HPA/I axis). In vertebrates, the urotensin-I group of orthologues, which includes sauvagine and urocortin, possess a number of shared characteristics that may be indicative of the ancestral peptide. Given the early origin of the CRF family peptides, it is likely that other peptide lineages are distantly related to the CRF family. In silico or cDNA library screening using probes based on urotensin-I/urocortin characteristics have been used to identify novel CRF family and related sequences that provide clues the evolutionary origin of the CRF family.     

Peptide inhibitors of HIV-1 protease and viral infection of peripheral blood lymphocytes based on HIV-1 Vif

We recently reported that HIV-1 Vif (virion infectivity factor) inhibits HIV-1 protease in vitro and in bacteria, suggesting that it may serve as the basis for the design of new protease inhibitors and treatment for HIV-1 infection. To evaluate this possibility, we synthesized peptide derivatives from the region of Vif, which inhibits protease, and tested their activity on protease. In an assay of cleavage of virion-like particles composed of HIV-1 Gag precursor polyprotein, full-length recombinant Vif, and a peptide consisting of residues 21–65 of Vif, but not a control peptide or BSA, inhibited protease activity. Vif_21–65 blocked protease at a molar ratio of two to one. We then tested this peptide and a smaller peptide, Vif_41–65, for their effects on HIV-1 infection of peripheral blood lymphocytes. Both Vif peptides inhibited virus expression below the limit of detection, but control peptides had no effect. To investigate its site of action, Vif_21–65 was tested for its effect on Gag cleavage by protease during HIV-1 infection. We found that commensurate with its reduction of virus expression, Vif_21–65 inhibited the cleavage of the polyprotein p55 to mature p24. These results are similar to those obtained by using Ro 31–8959, a protease inhibitor in clinical use. We conclude that Vif-derived peptides inhibit protease during HIV-1 infection and may be useful for the development of new protease inhibitors.     

A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins.

OBJECTIVES: Antibodies that detect coeliac-toxic prolamins from wheat, barley and rye are important tools for controlling the diet of coeliac disease patients. Recently, a monoclonal antibody R5 that recognizes wheat gliadin, barley hordein and rye secalin equally was described. In this study, the epitope recognized by R5 was investigated. METHODS: Both a phage-displayed heptapeptide library and overlapping peptides spanning the sequence of alpha- and gamma-type gliadins (pepscan) were screened for binding of R5. RESULTS: Both techniques yielded comparable pentapeptide consensus sequences (phage display QXPW/FP; pepscan QQPFP). According to recent observations, this peptide stretch may be of key importance in the pathogenicity of coeliac disease. This sequence occurs repetitively in prolamins (in gamma- and omega-type prolamins more frequently than in alpha-type prolamins) together with several homologous peptide stretches, which are recognized less strongly. CONCLUSIONS: R5 seems to be a good candidate for the specific detection of putative coeliac disease-active sequences in prolamins and thus represents a valuable tool for the quality control of gluten-free food.     

Screening for PreS specific binding ligands with a phage displayed peptides library

ABM: To construct a random peptide phage display library and search for peptides that specifically bind to the PreS region of hepatitis B virus (HBV). METHODS: A phage display vector, pFuse8, based on the gene 8 product (pⅧ) of M13 phage was made and used to construct a random peptide library. Ecoli derived thioredoxin-PreS was purified with Thio-bond beads, and exploited as the bait protein for library screening. Five rounds of bio-panning were performed. The PreS-binding specificities of enriched phages were characterized with phage ELISA assay. RESULTS: A phage display vector was successfully constructed as demonstrated to present a pⅧ fused HBV PreSl epitope on the phage surface with a high efficiency. A cysteine confined random peptide library was constructed containing independent clones exceeding 5±108 clone forming unit (CFU). A pool of phages showing a PreS-binding specificity was obtained after the screening against thio-PreS with an enrichment of approximately 400 times. Five phages with high PreS-binding specificities were selected and characterized. Sequences of the peptides displayed on these phages were determined. CONCLUSION: A phage library has been constructed, with random peptides displaying as pVIII-fusion proteins. Specific PreS-binding peptides have been obtained, which may be useful for developing antivirals against HBV infection.     

Peptide decoys selected by phage display block in vitro and in vivo activity of a human anti-FVIII inhibitor

Hemophilia A is a life-threatening, hemorrhagic, X-linked recessive disorder resulting in deficient factor VIII (FVIII) activity. After the infusion of therapeutic FVIII, 25% of patients develop anti-FVIII antibodies that inhibit FVIII procoagulant activity, thus precluding further administration of FVIII. Here we report a novel approach aimed at neutralizing the activity of FVIII inhibitors by peptide epitope surrogates. To illustrate our concept, we chose the human anti-FVIII monoclonal antibody, Bo2C11, as a representative of anti-FVIII antibodies and a phage-displayed peptide library approach to obtain surrogate peptides. We selected a series of constrained dodecapeptides with the core sequence W-NR, which specifically interacts with the combining site of Bo2C11. The peptides mimic the epitope recognized by Bo2C11 and are able to inhibit specifically and in a dose-dependent manner the binding of Bo2C11 to FVIII. Peptide 107, in particular, neutralized the activity of Bo2C11 in vitro and restored normal hemostasis in hemophilic mice. Thus, the use of peptide decoys may be a promising new approach for the neutralization of pathologic antibodies.     

Identification of the peptides that inhibit the function of human monoclonal thyroid-stimulating antibodies from phage-displayed peptide library.

Autoantibodies against TSH receptor (TSHR) are known to be involved in the occurrence of Graves' disease. It is obvious that mapping of epitopes of the autoantibodies found in the patients with Graves' disease is an important step in elucidating possible mechanism of generation of the autoantibodies against TSHR as well as in developing effective diagnostic and therapeutic approaches for Graves' disease. In this report we have identified the peptide sequences that bind to two human monoclonal thyroid-stimulating antibodies (mTSAbs; B6B7 and 101-2) from a disulfide-constrained phage-displayed peptide library. The peptides selected by three rounds of biopanning showed half-maximal inhibitory activities for cAMP synthesis induced by mTSAbs at about 0.1 micromol/L. SPWTLGA and TQWNMQH selected for B6B7 and 101-2, respectively, show specificity for their respective antibodies. This means that different clones of mTSAbs may have different epitopes for TSHR. The IgG of the patient from whom B6B7 was derived binds with specificity to the respective immobilized peptide in an enzyme-linked immunosorbant assay format, and its cAMP generation was also inhibited by selected peptide. It may be possible that the epitopes of TSAbs identified from the phage-displayed peptide library could be used for the classification of different clones of TSAbs present in patients with Graves' disease and for development of drugs to treat Graves' disease.     

Identification of highest-affinity ligands by affinity selection from equimolar peptide mixtures generated by robotic synthesis

A fully automated peptide synthesizer has been constructed that is capable of the simultaneous synthesis of up to 36 individual peptides and the synthesis of equimolar peptide mixtures. The instrument consists of an array of reaction vessels, a series of solenoid valves to control liquid flow, and a Zymark robot to deliver solvents and reagents; all components are computer controlled and coordinated. Equimolar peptide mixtures are obtained by algorithms that automate the mixing and distribution of peptide-resin particles. This technology was used to synthesize a library of 361 peptides, generated by randomizing two critical binding residues of a 10-mer epitope known to bind an anti-human immuno-deficiency virus gp120 monoclonal antibody. Each critical residue was substituted with 19 amino acids consisting of all the natural amino acids except cysteine. The library was synthesized as 19 pools, each containing 19 peptides. Each pool was screened in a solution-phase competition ELISA assay. The 12 most inhibitory peptides in the library were isolated by a rapid affinity-selection method and were identified by mass spectrometry and amino acid analysis. The binding properties of these 12 selected peptides were verified by synthesis and assay of the individual peptides. The two critical residues investigated were found to contribute independently to antibody binding.     

Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.     

Human recombinant single-chain antibody fragments, specific for the hypervariable region 1 of hepatitis C virus, from immune phage-display libraries

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) may contain a potential neutralization site and the generation of human single-chain antibody fragments (scFv) to HVR1 may therefore provide a useful tool for the study of HCV. In this report, we have isolated and characterized three anti-HVR1 scFv clones from two patient-derived phage-displayed libraries and HCV HVR1 peptides. scFv S52/20 and S53/6 were selected with serologically cross-reactive HVR1 peptides. scFv p3f10 was obtained by screening the library from patient MH with an autologous HVR1 peptide. Nucleotide sequencing showed that the V_H chains and V_κ chains of all three scFv antibodies were derived from V_H3 and Vκ1 family germline V-genes, respectively. The specificity and affinity of the recombinant scFv antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and an affinity biosensor, using HVR1 peptides. S52/20 scFv binding to S52 HVR1 peptide was blocked by preincubation with soluble peptide S52 and was partially competed by one of three HCV-infected patient sera. In addition, scFv S52/20 blocked the binding of HCV-susceptible Molt-4 cells to immobilized S52 peptide. This study demonstrates that recombinant human scFv antibodies to HCV HVR1 can be produced in vitro and directly confirms that HVR1 of HCV elicits highly specific antibodies. The very high specificity of these antibodies to HVR1 may limit their potential use in passive immunization therapy against HCV, and further engineering of the scFvs needs to be performed to generate broad-spectrum blocking scFvs.     

Identification of an additional negative regulatory region for p53 sequence-specific DNA binding

The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation. Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state. Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus. Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding. In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation. This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80–93 and 364–393. The C-terminal residues 364–393 are already well characterized as having negative regulatory function. DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80–93, defining this region as a previously unidentified negative regulatory domain of p53. Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80–93) are able to activate p53 sequence-specific DNA binding in vitro. We suggest that both negative regulatory regions, residues 80–93 and 364–393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.     

Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity

Development of cancer receptor-specific gold nanoparticles will allow efficient targeting/optimum retention of engineered gold nanoparticles within tumors and thus provide synergistic advantages in oncology as it relates to molecular imaging and therapy. Bombesin (BBN) peptides have demonstrated high affinity toward gastrin-releasing peptide (GRP) receptors in vivo that are overexpressed in prostate, breast, and small-cell lung carcinoma. We have synthesized a library of GRP receptor-avid nanoplatforms by conjugating gold nanoparticles (AuNPs) with BBN peptides. Cellular interactions and binding affinities (IC₅₀) of AuNP–BBN conjugates toward GRP receptors on human prostate cancer cells have been investigated in detail. In vivo studies using AuNP–BBN and its radiolabeled surrogate ¹⁹⁸AuNP–BBN, exhibiting high binding affinity (IC₅₀ in microgram ranges), provide unequivocal evidence that AuNP–BBN constructs are GRP-receptor-specific showing accumulation with high selectivity in GRP-receptor-rich pancreatic acne in normal mice and also in tumors in prostate-tumor-bearing, severe combined immunodeficient mice. The i.p. mode of delivery has been found to be efficient as AuNP–BBN conjugates showed reduced RES organ uptake with concomitant increase in uptake at tumor targets. The selective uptake of this new generation of GRP-receptor-specific AuNP–BBN peptide analogs has demonstrated realistic clinical potential in molecular imaging via x-ray computed tomography techniques as the contrast numbers in prostate tumor sites are severalfold higher as compared to the pretreatment group (Hounsfield unit = 150).     

A surface on the G protein β-subunit involved in interactions with adenylyl cyclases

Receptor activation of heterotrimeric G proteins dissociates Gα from the Gβγ complex, allowing both to regulate effectors. Little is known about the effector-interaction regions of Gβγ. We had used molecular modeling to dock a peptide encoding the region of residues 956–982 of adenylyl cyclase (AC) 2 onto Gβ to identify residues on Gβ that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86–105 (Gβ86–105) and 115–135 (Gβ115–135) from Gβ. The Gβ86–105 peptide inhibited Gβγ stimulation of AC2 and blocked Gβγ inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the Gβ115–135 peptide were similar to those of Gβ86–105 but Gβ115–135 was less efficacious in blocking Gβγ inhibition of AC1. Substitution of Tyr-124 with Val in the Gβ115–135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84–143 of Gβ as a surface that is involved in transmitting signals to effectors.