No abstract

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.

While the transcription-dependent mechanism of p53 has been extensively studied, recently the transcription-independent apoptotic activity of p53 has also been described. Bcl-2 and Bcl-X(L) interact with p53 and induce apoptosis. Initially, the p53 DNA-binding domain (p53DBD) was found to bind to Bcl-2 and Bcl-X(L). Later, the p53 N-terminal domain (p53NTD) was reported to be sufficient for inducing the transcription-independent apoptotic activity of p53 and also shown to interact with Bcl-X(L). Here, we further document that the transactivation domain of p53 (p53TAD) in p53NTD alone binds to Bcl-X(L). We demonstrated that the MDM2-binding region (residues S15 to N29, herein referred to as SN15) in p53TAD is the binding site for Bcl-X(L). The binding interface on Bcl-X(L) was identified at the hydrophobic pocket formed by the BH1, BH2, and BH3 domains, which also binds to the Bak/Bad BH3 peptides, suggesting Bcl-X(L) and MDM2 share a common binding motif in p53TAD. Our NMR structural studies have shown that the SN15 peptide undergoes a conformational change upon binding to Bcl-X(L). A Bcl-X(L)/SN15 complex structural model suggests that the SN15 peptide adopts an extended alpha-helical structure to bind to the hydrophobic pocket on the Bcl-X(L), which is similar to the mode of binding between BH3 peptides and Bcl-X(L).

This study describes the use of cyclic peptides for use in the selection of single-chain (ScFv) antibodies specific for the HIV-1 coreceptor CCR5, a representative G-protein-coupled receptor (GPCR). A tandem ligation strategy was developed for preparing biotinylated cyclic peptides, first through an orthogonal end-to-end ligation and then a chemoselective ligation with functionalized biotin. Cyclic peptides mimicking the extracellular loops of CCR5 and their unconstrained counterparts were then used for solution-phase selection of ScFv antibodies from a phage display antibody library. Antibodies reactive with CCR5 on cells were detected using a homogeneous high throughput assay. Of 19 isolated ScFv antibodies that bound to CCR5+ cells, three inhibited CCR5-mediated but not CXCR4-mediated HIV infection. Only ScFvs selected by binding to cyclic constrained peptides exhibited inhibitory activity. Our results demonstrate that surface-antigen mimetics of a GPCR are effective tools for selecting active, site-specific ScFv antibodies that hold promise as immunological reagents and therapeutics.

The assembly of synthetic peptide-based vaccines that incorporate multiple epitopes is a major goal of vaccine development, because such vaccines will potentially allow the immunization of outbred populations against a number of different pathogens. We have shown that free radical-induced polymerization of individual peptide epitopes results in the incorporation of multiple copies of the same or different epitopes into high molecular weight immunogens (O'Brien-Simpson, N.M., Ede, N.J., Brown, L.E., Swan, J. & Jackson, D.C. (1997) Polymerization of unprotected synthetic peptides: a view toward synthetic peptide vaccines. J. Am. Chem. Soc.119, 1183-1188; Jackson, D.C., O'Brien-Simpson, N., Ede, N.J. & Brown, L.E. (1997) Free radical induced polymerization of synthetic peptides into polymeric immunogens. Vaccine 15, 1697-1705). The ability to control the size of these polymers, to determine the physical and chemical properties of the backbone material and also to know the extent to which individual peptide epitopes are incorporated are important manufacturing considerations and form the subject of this study. We show here that the polymerization process is highly efficient with at least 70% of peptides incorporated into the resulting polymer, that acrylamide and acryloylated amino acids can be used as comonomers with peptide epitopes in the polymerization reaction and that the choice of the comonomer can influence the properties of the resulting polymer. We also show that the size of chain growth polymers is restricted in the presence of chain transfer agents, that the resulting polymer size can be predicted and that there is little or no difference in the immunogenicity of polymers that range in apparent molecular size between 18 kDa and 335 kDa. The successful polymerization of peptide epitopes with an acryloyl-amino acid creates the potential for introducing different physical and chemical properties into artificial protein immunogens.