Modulating angiogenesis is an attractive goal because many pathological conditions depend on the growth of new vessels. Angiogenesis is mainly regulated by the VEGF, a mitogen specific for endothelial cells. In the last years, many efforts have been pursued to modulate the angiogenic response targeting VEGF and its receptors. Based on the x-ray structure of VEGF bound to the receptor, we designed a peptide, QK, reproducing a region of the VEGF binding interface: the helix region 17-25. NMR conformation analysis of QK revealed that it adopts a helical conformation in water, whereas the peptide corresponding to the ␣-helix region of VEGF, VEGF15, is unstructured. Biological assays in vitro and on bovine aorta endothelial cells suggested that QK binds to the VEGF receptors and competes with VEGF. VEGF15 did not bind to the receptors indicating that the helical structure is necessary for the biological activity. Furthermore, QK induced endothelial cells proliferation, activated cell signaling dependent on VEGF, and increased the VEGF biological response. QK promoted capillary formation and organization in an in vitro assay on matrigel. These results suggested that the helix region 17-25 of VEGF is involved in VEGF receptor activation. The peptide designed to resemble this region shares numerous biological properties of VEGF, thus suggesting that this region is of potential interest for biomedical applications, and molecules mimicking it could be attractive for therapeutic and diagnostic applications.helical peptides ͉ peptide design ͉ VEGF ͉ angiogenesis ͉ NMR spectroscopy
Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton. They bind DNA and RNA with high specificity and selectivity, leading to PNA-RNA and PNA-DNA hybrids more stable than the corresponding nucleic acid complexes. The binding affinity and selectivity of PNAs for nucleic acids can be modified by the introduction of stereogenic centers (such as D-Lys-based units) into the PNA backbone. To investigate the structural features of chiral PNAs, the structure of a PNA decamer containing three D-Lys-based monomers (namely H-GpnTpnApnGpnAdlTdlCdlApnCpnTpn-NH2, in which pn represents a pseudopeptide link and dl represents a D-Lys analogue) hybridized with its complementary antiparallel DNA has been solved at a 1.66-Å resolution by means of a single-wavelength anomalous diffraction experiment on a brominated derivative. The D-Lys-based chiral PNA-DNA (LPD) heteroduplex adopts the so-called P-helix conformation. From the substantial similarity between the PNA conformation in LPD and the conformations observed in other PNA structures, it can be concluded that PNAs possess intrinsic conformational preferences for the P-helix, and that their flexibility is rather restricted. The conformational rigidity of PNAs is enhanced by the presence of the chiral centers, limiting the ability of PNA strands to adopt other conformations and, ultimately, increasing the selectivity in molecular recognition. P eptide nucleic acids (PNAs) are oligonucleotide mimics in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton, composed of N-(2-aminoethyl)glycine units (1) (Fig. 1). Nucleobases are linked to this skeleton through a two-atom carboxymethyl spacer.PNAs bind DNA and RNA with high specificity and selectivity, forming Watson-Crick base pairs and leading to PNA-RNA and PNA-DNA hybrids that are more stable than the corresponding nucleic acid complexes (2). Because of their high thermal stability and resistance to proteases and nucleases, PNAs are ideal candidates as antisense or antigene therapeutic agents (3-6) and are currently used as powerful tools in molecular biology and in diagnostics (7).Three-dimensional structures have been determined for the major families of PNA complexes by different techniques. A PNA-RNA duplex (8) and a PNA-DNA duplex (9) were characterized by NMR in solution, whereas a (PNA) 2 -DNA triplex (10) and three PNA-PNA duplexes (11-13) were solved by x-ray crystallography. The structural analysis in solution of the PNA-DNA (9) and PNA-RNA duplexes (8) showed that PNA, when hybridized to RNA, adopts an A-like helix, whereas, when hybridized to a complementary DNA strand, it adopts a conformation that is different from both the A and the B forms. The crystal structure of the (PNA) 2 -DNA triplex (10) also showed helical parameters significantly different from those of canonical DNA or RNA helical forms, defining a type of helix, named the P-helix, characterized by a small twist angle, a large x-displacem...
Cationic antimicrobial peptides (AMPs) possess fast and broad-spectrum activity against both Gram-negative and Gram-positive bacteria, as well as fungi. It has become increasingly evident that many AMPs, including those that derive from fragments of host proteins, are multifunctional and able to mediate various immunomodulatory functions and angiogenesis. Among these, synthetic apolipoprotein-derived peptides are safe and well tolerated in humans and have emerged as promising candidates in the treatment of various inflammatory conditions. Here, we report the characterization of a new AMP corresponding to residues 133-150 of human apolipoprotein E. Our results show that this peptide, produced either by chemical synthesis or by recombinant techniques in Escherichia coli, possesses a broad-spectrum antibacterial activity. As shown for several other AMPs, ApoE (133-150) is structured in the presence of TFE and of membrane-mimicking agents, like SDS, or bacterial surface lipopolysaccharide (LPS), and an anionic polysaccharide, alginate, which mimics anionic capsular exo-polysaccharides of several pathogenic microorganisms. Noteworthy, ApoE (133-150) is not toxic toward several human cell lines and triggers a significant innate immune response, assessed either as decreased expression levels of proinflammatory cytokines in differentiated THP-1 monocytic cells or by the induction of chemokines released from PBMCs. This novel bioactive AMP also showed a significant anti-inflammatory effect on human keratinocytes, suggesting its potential use as a model for designing new immunomodulatory therapeutics.
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