Suppression of Membrane Phospholipase C Activity by Synthetic Autoinhibitor Peptides

It has been demonstrated that the phospholipase C-γ (PLCγ) molecule possesses within it a phospholipase C inhibitor (PCI) region and that synthetic peptides based on the sequence of the PCI region suppress the enzymatic activity of PLC isoforms [Y. Homma and T. Takenawa (1992) J. Biol. Chem.267, 21884–218891. For the present study, the applicability of these peptides as inhibitors of PLC activity In plasma membranes was examined. Synthetic peptides, the original PCI peptide (24-mer) and a minimum octamer (YRKMRLRY), inhibit Ca²⁺-inducible PLC activation in digitonine-permeabilized cells, while a dodecamer sequence within the PCI region (SYYEKHALYRKM) does not. Similar results were obtained in both agonist- and GTP-binding protein-inducible PLC activation systems using purified plasma membranes. The inhibitory effect described here appears to reflect the inhibitory potency of the peptides against purified PLC isoforms. Therefore, these inhibitor peptides could provide an excellent tool for analyzing protein–protein interactions and resulting PLC activation.     

Inhibition of antigen-induced mediator release from IgE-sensitized cells by a monoclonal anti-Fc epsilon RI alpha-chain receptor antibody: implications for the involvement of the membrane-proximal alpha-chain region in Fc epsilon RI-mediated cell activation

The interaction between human IgE and its high affinity receptor, FcepsilonRI, is a critical event in mediating the allergic response. Aggregation of the alpha-chain of FcepsilonRI (FcepsilonRIalpha) occurs via cross-linking of receptor-bound IgE by Ag, resulting in cell activation and the release of mediators of hypersensitivity. Recently, we mapped the epitopes of two anti-FcepsilonRIalpha mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcepsilonRIalpha. Here we demonstrate both 5H5F8 binding to FcepsilonRI(+) cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca(2+) flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcepsilonRIalpha. Further, 5H5F8 and its Fab inhibit sulfidoleukotriene and histamine release from primary human peripheral blood leukocytes, including cells bearing endogenous IGE: Furthermore, we confirm that 5H5F8 maps to a linear peptide sequence in close proximity to the cell membrane. Two chemically synthesized peptides containing the 5H5F8 epitope sequence PREKY were selected for detailed analysis of 5H5F8 and 5H5F8 Fab binding and were found to produce K(d) values of similar magnitude to that observed for binding to recombinant FcepsilonRIalpha. These peptides may prove useful as targets for the identification of antagonists of FcepsilonRIalpha-mediated biological activity. Moreover, our data indicate that FcepsilonRIalpha-mediated activation may involve a novel alpha-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.     

SH2 domain-containing inositol polyphosphate 5'-phosphatase is the main mediator of the inhibitory action of the mast cell function-associated antigen.

The mast cell function-associated Ag (MAFA) is a type II membrane glycoprotein originally found on the plasma membrane of rat mucosal-type mast cells (RBL-2H3 line). A C-type lectin domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM) are located in the extracellular and intracellular domains of MAFA, respectively. MAFA clustering has previously been shown to suppress the secretory response of these cells to the FcepsilonRI stimulus. Here we show that the tyrosine of the ITIM undergoes phosphorylation, on MAFA clustering, that is markedly enhanced on pervanadate treatment of the cells. Furthermore, the Src homology 3 domain of the protein tyrosine kinase Lyn binds directly to a peptide containing nonphosphorylated MAFA ITIM and PAAP motif. Results of both in vitro and in vivo experiments suggest that Lyn is probably responsible for this ITIM phosphorylation, which increases the Src homology domain 2 (SH2) affinity of Lyn for the peptide. In vitro measurements established that tyrosine-phosphorylated MAFA ITIM peptides also bind the SH2 domains of inositol 5'-phosphatase (SHIP) as well as protein tyrosine phosphatase-2. However, the former single domain is bound 8-fold stronger than both of the latter. Further support for the role of SHIP in the action of MAFA stems from in vivo experiments in which tyrosine-phosphorylated MAFA was found to bind primarily SHIP. In RBL-2H3 cells overexpressing wild-type SHIP, MAFA clustering causes markedly stronger inhibition of the secretory response than in control cells expressing normal SHIP levels or cells overexpressing either wild-type protein tyrosine phosphatase-2 or its dominant negative form. In contrast, on overexpression of the SH2 domain of SHIP, the inhibitory action of MAFA is essentially abolished. Taken together, these results suggest that SHIP is the primary enzyme responsible for mediating the inhibition by MAFA of RBL-2H3 cell response to the FcepsilonRI stimulus.     

Identification of active peptide sequences in the carboxyl-terminal cell binding domain of human thrombospondin-1.

Thrombospondin (TS) mediates attachment, spreading, and motility of several cell types through at least four cell binding domains: the amino-terminal heparin binding domain, the type I repeats containing the CSVTCG sequence, the RGDA sequence in the last of the type III calcium binding repeats and the carboxyl-terminal cell or platelet binding domain (CBD). The attachment of human melanoma cells (G361) to the COOH-terminal domain is independent of the RGDA sequence and is inhibited by the monoclonal antibody C6.7. To define the cell binding site(s) within this 212-residue COOH-terminal domain, we have synthesized eight overlapping peptides (seven 30-mers and a final 37-mer) representing the entire sequence of the CBD. Several of these peptides are insoluble in aqueous buffers at high concentration. Cell adhesion assays have been devised which employ covalent coupling of peptides in chaotropic solvents to chemically derivatized plastic 96-well plates. Three synthetic peptides, two of which are nonadjacent in the linear sequence, are potent attachment factors for G361 cells. C6.7 blocks adhesion to one of these peptides, whereas sulfated glycoconjugates inhibit adhesion of cells to all three. Polyclonal antibodies raised against the peptides inhibit cell adhesion to the peptides, the recombinant CBD, and to intact TS. The peptides GRGDSP and VTCG are not inhibitory. These sites are thus independent from the type I repeats and the RGDA sequence of TS. Each of the active peptides inhibits cell attachment to the other active peptides as well as to the CBD and to intact TS. This mutual inhibition suggests that the peptides share a common cellular receptor which may contain an associated glycoconjugate chain. These data indicate that the COOH-terminal cell binding domain of TS contains at least two peptide sequences which contribute to the attachment of a wide variety of cells.     

TGF-beta 1 inhibits mast cell Fc epsilon RI expression

Mast cell activation through the high affinity IgE receptor (FcepsilonRI) is a critical component of atopic inflammation. The cytokine TGF-beta1 has been shown to inhibit IgE-dependent mast cell activation, possibly serving to dampen mast cell-mediated inflammatory responses. We present proof that TGF-beta1 inhibits mast cell FcepsilonRI expression through a reversible pathway that diminishes protein, but not mRNA, expression of the FcepsilonRI subunit proteins alpha, beta, and gamma. The stability of the expressed proteins and the assembled cell surface complex was unaltered by TGF-beta1 treatment. However, TGF-beta1 decreased the rate of FcepsilonRI beta-chain synthesis, arguing that this inhibitory cytokine exerts its effects at the level of mRNA translation. TGF-beta1 consistently diminished FcepsilonRI expression on cultured human or mouse mast cells as well as freshly isolated peritoneal mast cells. The related cytokines, TGF-beta2 and TGF-beta3, had similar effects. We propose that TGF-beta1 acts as a negative regulator of mast cell function, in part by decreasing FcepsilonRI expression.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Convergent recognition of the IgE binding site on the high-affinity IgE receptor

Two structurally distinct classes of peptides were recently identified by phage display that bind the high-affinity IgE receptor, FcepsilonRI, and block IgE binding and subsequent receptor activation. Both classes adopt highly stable structures in solution, one forming a beta hairpin, with the other forming a helical "zeta" structure. Despite these differences, the two classes bind competitively to the same site on the receptor. Structural analyses of both peptide-receptor complexes by NMR spectroscopy and/or X-ray crystallography reveal that the unrelated peptide scaffolds have nevertheless converged to present a similar three-dimensional surface to interact with FcepsilonRI and that their modes of interaction share a key feature of the IgE-FcepsilonRI complex, the proline/tryptophan sandwich.     

Mode of action of two inhibitory peptides from heptad repeat domains of the fusion protein of Newcastle disease virus

Peptides derived from heptad repeat (HR) sequences of viral fusion proteins from several enveloped viruses have been shown to inhibit virus-mediated membrane fusion but the mechanism remains unknown. To further investigate this, the inhibition mechanism of two HR-derived peptides from the fusion protein of the paramyxovirus Newcastle disease virus (NDV) was investigated. Peptide N24 (residues 145-168) derived from HR1 was found to be 145-fold more inhibitory in a syncytium assay than peptide C24 (residues 474-496), derived from HR2. Both peptides failed to block lipid-mixing between R18-labeled virus and cells. None of the peptides interfered with the binding of hemagglutinin-neuraminidase (HN) protein to the target cells, as demonstrated by hemagglutining assays. When both peptides were mixed at equimolar concentrations, their inhibitory effect was abolished. In addition, both peptides induced the aggregation of negatively charged and zwitterionic phospholipid membranes. The ability of the peptides to interact with each other in solution suggests that these peptides may bind to the opposite HR region on the protein whereas their ability to interact with membranes as well as their failure to block lipid transfer suggest a second binding site. Taken together these results, suggest a mode of action for C24 and N24 in which both peptides have two different targets on the F protein: the opposite HR sequence and their corresponding domains.     

MHC class II binding of peptides derived from HLA-DR 1.

The aim of these studies was to determine whether auto- and alloreactivity can arise from T cell recognition of MHC-peptides in context of syngeneic MHC. Four synthetic peptides derived from the first domain of the HLA-DR beta 1 * 0101 chain were used in limiting dilution analysis to prime T cells from HLA-DR1- and HLA-DR1+ responders. The frequency of T cells responding to these four peptides was similar in individuals with or without HLA-DR1. In both cases, the peptide corresponding to the nonpolymorphic sequence 43-62, was less immunogenic than peptides corresponding to the three hypervariable regions 1-20, 21-42, and 66-90, eliciting a lower number of reactive T cells. Experiments using a T cell line with specific reactivity to peptide 21-42 showed, however, that this response can be efficiently blocked by adding to the culture a nonpolymorphic sequence peptide. This suggests that alloreactivity can be blocked by use of monomorphic (self) peptides. The binding of both "monomorphic" and "polymorphic" synthetic DR1 peptides to affinity purified HLA-DR 1 and DR 11 molecules was measured using radiolabeled peptides and high performance size exclusion chromatography. The data showed that the polymorphic as well as monomorphic synthetic DR1 peptides bound to both DR1 and DR11 molecules. Competitive inhibition studies indicated that the monomorphic 43-62 peptide can block the binding of the polymorphic peptides, consistent with the results obtained in T cell cultures. Taken together these data suggest that anti-MHC autoreactive T cells are present in the periphery and that both auto and alloreactivity can be elicited by MHC peptides binding to MHC class II molecules.     

Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen

The alpha 2 beta 1 integrin serves as either a specific cell surface receptor for collagen or as both a collagen and laminin receptor depending upon the cell type. Recently we established that the alpha 2 beta 1 integrin binds to a site within the alpha 1 (I)-CB3 fragment of type I collagen (Staatz, W. D., Walsh, J. J., Pexton, T., and Santoro, S. A. (1990) J. Biol. Chem. 265, 4778-4781). To define the alpha 2 beta 1 recognition sequence further we have prepared an overlapping set of synthetic peptides which completely spans the 148-amino acid alpha 1(I)-CB3 fragment and tested the peptides for ability to inhibit cell adhesion to collagen and laminin substrates. The minimal active recognition sequence defined by these experiments is a tetrapeptide of the sequence Asp-Gly-Glu-Ala (DGEA) corresponding to residues 435-438 of the type I collagen sequence. The DGEA-containing peptides effectively inhibited alpha 2 beta 1-mediated Mg2(+)-dependent adhesion of platelets, which use the alpha 2 beta 1 integrin as a collagen-specific receptor, to collagen but had no effect on alpha 5 beta 1-mediated platelet adhesion to fibronectin or alpha 6 beta 1-mediated platelet adhesion to laminin. In contrast, with T47D breast adenocarcinoma cells, which use alpha 2 beta 1 as a collagen/lamin receptor, adhesion to both collagen and laminin was inhibited by DGEA-containing peptides. Deletion of the alanine residue or substitution of alanine for either the glutamic or aspartic acid residues in DGEA-containing peptides resulted in marked loss of inhibitory activity. These results indicate that the amino acid sequence DGEA serves as a recognition site for the alpha 2 beta 1 integrin complex on platelets and other cells.     

Peptide boronic acids, substrate analogs, inhibit chymase, and histamine release from rat mast cells

Peptide boronic acids, such as methoxysuccinyl-Ala-Ala-Pro-(L)boro-Phe-OH, its pinacol ester, and t-butyloxycarbonyl-Phe-Pro-(L)boro-Phe-pinacol, inhibited the activity of chymase from connective tissue mast cells approximately 40- to 80-fold more than atypical chymase from mucosal mast cells, and did not inhibit trypsin. Only peptide boronic acids containing "L" forms of boronic acids were inhibitory. The Ki values of these peptide boronic acids for chymase were in the 60-170 nM concentration range, like those of the natural inhibitors tested, but all the natural inhibitors tested except Eglin C and chymostatin inhibited both chymase and trypsin. Thus these peptide boronic acids should be useful for selective inhibition of chymase with less inhibitory activity for atypical chymase and without inhibition of trypsin. These peptide boronic acids markedly inhibited histamine release induced by anti-rat immunoglobulin E, suggesting that chymase in connective tissue mast cells plays some role in the process of histamine release. These peptides are assumed to be therapeutically useful for treatment of allergic inflammations catalyzed by chymase.     

Peptide inhibitors of fibronectin, laminin, and other adhesion molecules: unique and shared features

Synthetic peptides can specifically inhibit the function of certain adhesive glycoproteins in vitro and in vivo. We have compared the relative activities of a set of six variant synthetic peptides based on the sequence of fibronectin in terms of their ability to inhibit the interactions of fibroblasts with fibronectin, spreading factor/vitronectin, laminin, and native collagen gels. BHK (baby hamster kidney) and chick embryo fibroblasts spreading on these adhesive molecules displayed distinctive patterns of sensitivity to inhibition by this panel of peptides, which depended on the adhesive molecule rather than the cell type. For fibronectin, Gly-Arg-Gly-Asp-Ser was considerably more active than Arg-Gly-Asp-Ser, whereas these two peptides displayed little difference in activity in inhibiting cell adhesion to spreading factor. For both proteins, the inverted peptide sequence Ser-Asp-Gly-Arg was also moderately active, whereas closely related peptides containing a transposition, a deletion, or a single, conserved amino acid substitution were much less active. For inhibiting interactions with laminin or native type I collagen gels, Gly-Arg-Gly-Asp-Ser was only weakly active, but the inverted peptide Ser-Asp-Gly-Arg unexpectedly continued to display inhibitory activity for both attachment proteins in both cell types. Our results indicate that different adhesive processes depend on distinct peptide recognition events by a cell. However, there may be a possible common denominator among attachment proteins in a moderate sensitivity to Ser-Asp-Gly-Arg. Our study also underscores the importance of examining a full set of peptide analogs when these novel inhibitors are used to characterize biological processes.     

An inhibitory mechanism of action of coiled‐coil peptides against type three secretion system from enteropathogenic Escherichia coli

Human pathogenic gram‐negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled‐coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha‐helical structures that play an important role in the protein‐protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled‐coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS‐dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking‐molecular dynamic simulations, and quantum‐mechanic calculations of the various peptide‐protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled‐coil domain of the C‐terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide‐protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad‐spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern.     

A discontinuous factor H binding site in the third component of complement as delineated by synthetic peptides

Factor H, a very important regulator of alternative pathway activation, exerts its effects by binding to the third component complement, C3. In this study we present evidence that factor H reacts with at least two sites in the third component of complement (C3), and we have mapped one of these sites within the C3d fragment of C3. By using direct binding assays of an anti-human H anti-idiotypic antibody (alpha alpha H) and of H to C3 fragments, it was shown that both bound to the C3b and C3d (but not to C3c) fragments of C3. Cleavage of C3d by CNBr generated two major fragments with Mr values of 12,500 (residues 997-1107) and 8,600 (residues 1178-1252). Binding studies with these two fragments showed that only the Mr 8,600 fragment bound to both H and alpha alpha H. Several synthetic peptides (A58, 1192-1249; P28, 1187-1214; P16, 1194-1209; P14, 1201-1214; B17, 1206-1222; J28, 1222-1249; and J16, 1234-1249) were synthesized according to the primary sequence of the Mr 8,600 fragment. Based on the differential binding of these synthetic peptides to H, their inhibitory effect on H binding to C3b or C3d, and their effect on H cofactor activity, we mapped the H binding site in C3 to a discontinuous site spanning residues 1187-1249 of the C3 sequence. By studying the inhibition of H binding to C3b or C3d by the different synthetic peptides, we also present evidence that a second binding site in C3b for H exists.     

Peptides derived from type I thrombospondin repeat-containing proteins of the CCN family inhibit proliferation and migration of endothelial cells

Angiogenesis, or neovascularization, is tightly orchestrated by endogenous regulators that promote or inhibit the process. The fine-tuning of these pro- and anti-angiogenic elements (the angiogenic balance) helps establish the homeostasis in tissues, and any aberration leads to pathologic conditions. The type I thrombospondin repeats are a family of protein structural elements involved in the control of angiogenesis, and some proteins containing these repeats have been identified as negative regulators of angiogenesis. Here we identify a set of 11 novel, anti-angiogenic 18–20-amino acid peptides that are derived from proteins that belong to the CCN protein family and contain type I thrombospondin motifs. We have named these peptides spondinstatin-1, cyrostatin, connectostatin, nephroblastostatin, wispostatin-2, wispostatin-3, netrinstatin-5C, netrinstatin-5D, adamtsostatin-like-4, fibulostatin-6.1, and complestatin-C6 to reflect their origin. We have shown that these peptides inhibit proliferation and migration of human umbilical vein endothelial cells in vitro. By conducting a clustering analysis of the amino acid sequences using sequence similarity criteria and of the experimental results using a hierarchical clustering algorithm, we have demonstrated that there is an underlying correlation between the sequence and activity of the identified peptides. This combination of experimental and computational approaches introduces a novel systematic framework for studying peptide activity, identifying novel peptides with anti-angiogenic activity, and designing mimetic peptides with tailored properties.     

Lipid raft-associated catechin suppresses the FcepsilonRI expression by inhibiting phosphorylation of the extracellular signal-regulated kinase1/2

The major green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has a suppressive effect on the expression of the high-affinity IgE receptor FcepsilonRI, which is key molecule in the IgE-mediated allergic reactions. Here we show that EGCG binds to the cell surface and highly associates with plasma membrane microdomains, lipid rafts, on the human basophilic KU812 cells. The disruption of these lipid rafts caused a reduction of the amount of raft-associated EGCG and the FcepsilonRI-suppressive effect of EGCG. We also found that EGCG has an ability to inhibit the phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2) and that the ERK1/2 specific inhibitor also reduced FcepsilonRI expression. Moreover, the inhibitory effect elicited by EGCG on ERK1/2 was prevented by disruption of rafts. Thus, these results suggest that the interaction between EGCG and the lipid rafts is important for EGCG's ability to downregulate FcepsilonRI expression, and ERK1/2 may be involved in this suppression signal.     

The use of peptide analogs with improved stability and MHC binding capacity to inhibit antigen presentation in vitro and in vivo

The identification of a core region for OVA 323-339, which is critical in determining binding to IAd, has enabled us to generate a series of analog peptides in which this core region was extended at both the N and C termini with different amino acid residues. When assessed for binding capacity, several peptides were shown to have increased affinity for IAd compared with the parent sequence, and in addition, some peptides had acquired binding specificities for class II MHC haplotypes not present for OVA 323-339. These peptides were next examined for their ability to inhibit T cell responses in vitro and in vivo. The correlation between binding and the ability to inhibit T cell activation in vitro was good. However, when assessed in vivo, it was clear that high Ia binding was not sufficient in itself to define the inhibitory capacity of a given peptide. That this discrepancy was due to differences in degradation of the core-extended peptides was suggested by 1) results from an inhibition of Ag presentation assay, in which the pulse period with Ag and inhibitor was extended to 20 h; and 2) direct analysis of peptide stability by using reverse phase HPLC. Finally, by protecting the peptide from degradation with N- and C-terminal substitutions of D-amino acids, the inhibitory capacity of an unstable core-extended peptide in vitro could be greatly enhanced. These data indicate that the core extension approach may be one method by which antagonists for MHC class II molecules may be generated.     

Development of a functional backbone cyclic mimetic of the HIV-1 Tat arginine-rich motif

We have used the backbone cyclic proteinomimetics approach to develop peptides that functionally mimic the arginine-rich motif (ARM) of the HIV-1 Tat protein. This consensus sequence serves both as a nuclear localization signal (NLS) and as an RNA binding domain. Based on the NMR structure of Tat, we have designed and synthesized a backbone cyclic ARM mimetic peptide library. The peptides were screened for their ability to mediate nuclear import of the corresponding BSA conjugates in permeabilized cells. One peptide, designated "Tat11," displayed active NLS properties. Nuclear import of Tat11-BSA was found to proceed by the same distinct pathway used by the Tat-NLS and not by the common importin alpha pathway, which is used by the SV40-NLS. Most of the Tat-derived backbone cyclic peptides display selective inhibitory activity as demonstrated by the inhibition of the nuclear import mediated by the Tat-NLS and not by the SV40-NLS. The Tat-ARM-derived peptides, including Tat-11, also inhibited binding of the HIV-1 Rev-ARM to its corresponding RNA element (Rev response element) with inhibition constants of 5 nm. Here we have shown for the first time (a) a functional mimetic of a protein sequence, which activates a nuclear import receptor and (b) a mimetic of a protein sequence with a dual functionality. Tat11 is a lead compound which can potentially inhibit the HIV-1 life cycle by a dual mechanism: inhibition of nuclear import and of RNA binding.     

Thrombin inhibition by cyclic peptides from thrombomodulin.

Peptides corresponding to the loop regions of the fourth, fifth, and sixth epidermal growth factor (EGF)-like domains of thrombomodulin (TM) have been synthesized and assayed for thrombin inhibition, as indicated by both inhibition of thrombin-mediated fibrinogen clotting and inhibition of the association of thrombin with TM that results in protein C activation. Peptides from the fifth EGF-like domain showed significant inhibition of fibrinogen clotting and protein C activation, whereas peptides from the fourth and sixth EGF-like domains were weak inhibitors in both assays. Two structural features were important for inhibitory potency of the peptides from the fifth EGF-like domain: cyclization by a disulfide bond and attachment of the "tail" amino acids C-terminal to the disulfide loop. Linear control peptides did not significantly inhibit clotting or protein C activation. The C-terminal loop alone, the "tail" peptide, or a mixture of the two were at least 10-fold less potent inhibitors of clotting or protein C activation. A more constrained peptide analog was designed by deletion of an isoleucine within the C5-C6 disulfide loop, TM52-1 + 5C. This analog was a better inhibitor in both assay systems, having a Ki for protein C activation of 26 microM.     

Anti-complement effects of lactoferrin-derived peptides

Lactoferrin is an important biological molecule with many functions such as modulation of the inflammatory response, iron metabolism and antimicrobial defense. One effect of lactoferrin is the inhibition of the classical complement pathway. This study reports that antimicrobial peptides derived from the N-terminal region from both human and bovine lactoferrin, lactoferricin H and lactoferricin B, respectively, inhibit the classical complement pathway. No inhibitory effect of these peptides was observed on the alternative complement pathway in an AP50 assay. However, lactoferricin B reduced the inhibitory properties of serum against Escherichia coli in a concentration dependent manner. These results suggest that the N-terminal region of lactoferrin is the important part in the inhibition of complement activation and that these peptides possess other important properties than their antimicrobial effect.