Domenica Capasso

Insights into peptide nucleic acid (PNA) structural features: The crystal structure of a D-lysine-based chiral PNA-DNA duplex

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton.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. Thed-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.

Functional and pharmacological characterization of a VEGF mimetic peptide on reparative angiogenesis

Vascular endothelial growth factor (VEGF) is the main regulator of physiological and pathological angiogenesis. Low molecular weight molecules able to stimulate angiogenesis have interesting medical application for example in regenerative medicine, but at present none has reached the clinic. We reported that a VEGF mimetic helical peptide, QK, designed on the VEGF helix sequence 17-25, is able to bind and activate the VEGF receptors, producing angiogenesis. In this study we evaluate the pharmacological properties of peptide QK with the aim to propose it as a VEGF-mimetic drug to be employed in reparative angiogenesis. We show that the peptide QK is able to recapitulate all the biological activities of VEGF in vivo and on endothelial cells. In experiments evaluating sprouting from aortic ring and vessel formation in an in vivo angiogenesis model, the peptide QK showed biological effects comparable with VEGF. At endothelial level, the peptide up-regulates VEGF receptor expression, activates intracellular pathways depending on VEGFR2, and consistently it induces endothelial cell proliferation, survival and migration. When added to angiogenic factors (VEGF and/or FGF-2), QK produces an improved biological action, which resulted in reduced apoptosis and accelerated in vitro wound healing. The VEGF-like activity of the short peptide QK, characterized by lower cost of production and easier handling compared to the native glycoprotein, suggests that it is an attractive candidate to be further developed for application in therapeutic angiogenesis.

Role of Surface-Exposed Loops of Haemophilus influenzae Protein P2 in the Mitogen-Activated Protein Kinase Cascade

ABSTRACT The outer membrane of gram-negative bacteria contains several proteins, and some of these proteins, the porins, have numerous biological functions in the interaction with the host; porins are involved in the activation of signal transduction pathways and, in particular, in the activation of the Raf/MEK1-MEK2/mitogen-activated protein kinase (MAPK) cascade. The P2 porin is the most abundant outer membrane protein of Haemophilus influenzae type b. A three-dimensional structural model for P2 was constructed based on the crystal structures of Klebsiella pneumoniae OmpK36 and Escherichia coli PhoE and OmpF. The protein was readily assembled into the β-barrel fold characteristic of porins, despite the low sequence identity with the template proteins. The model provides information on the structural features of P2 and insights relevant for prediction of domains corresponding to surface-exposed loops, which could be involved in the activation of signal transduction pathways. To identify the role of surface-exposed loops, a set of synthetic peptides were synthesized according to the proposed model and were assayed for MEK1-MEK2/MAPK pathway activation. Our results show that synthetic peptides corresponding to surface loops of protein P2 are able to activate the MEK1-MEK2/MAPK pathways like the entire protein, while peptides modeled on internal β strands are unable to induce significant phosphorylation of the MEK1-MEK2/MAPK pathways. In particular, the peptides corresponding to loops L5 (Lys206 to Gly219), L6B (Ser239 to Lys253), and L7 (Thr280 to Lys287) activate, as the whole protein, essentially JNK and p38.

γ Sulphate PNA (PNA S): highly selective DNA binding molecule showing promising antigene activity.

Peptide Nucleic Acids (PNAs), nucleic acid analogues showing high stability to enzyme degradation and strong affinity and specificity of binding toward DNA and RNA are widely investigated as tools to interfere in gene expression. Several studies have been focused on PNA analogues with modifications on the backbone and bases in the attempt to overcome solubility, uptake and aggregation issues. γ PNAs, PNA derivatives having a substituent in the γ position of the backbone show interesting properties in terms of secondary structure and affinity of binding toward complementary nucleic acids. In this paper we illustrate our results obtained on new analogues, bearing a sulphate in the γ position of the backbone, developed to be more DNA-like in terms of polarity and charge. The synthesis of monomers and oligomers is described. NMR studies on the conformational properties of monomers and studies on the secondary structure of single strands and triplexes are reported. Furthermore the hybrid stability and the effect of mismatches on the stability have also been investigated. Finally, the ability of the new analogue to work as antigene, interfering with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), assessed by FACS and qPCR, is described.

RNA-Binding and Viral Reverse Transcriptase Inhibitory Activity of a Novel Cationic Diamino Acid-Based Peptide

A novel cationic peptide based on L-lysine and L-diaminobutyric acid was prepared for the first time by solid phase synthesis. After HPLC purification and ESI MS characterization, we studied by CD and IR spectroscopy the structural features of the novel basic peptide, which is able to form a β-turn-like structure. Furthermore, its interaction with DNA and RNA was investigated by CD and UV spectroscopy, which revealed a preferential RNA-binding ability of the sequential peptide, whereas its inhibitory activity toward HIV and Moloney murine leukemia virus (MMLV) reverse transcriptase action was evaluated by semiquantitative PCR. The cationic sequential peptide was able to inhibit the reverse transcriptase activity in both cases, even if our PCR data suggested a major activity in the case of HIV-RT, probably due to the stronger cationic peptide-protein interaction evidenced by UV spectroscopy.

gH625 is a viral derived peptide for effective delivery of intrinsically disordered proteins

A genetically modified recombinant gH625-c-prune was prepared through conjugation of c-prune with gH625, a peptide encompassing 625–644 residues of the glycoprotein H of herpes simplex virus 1, which has been proved to possess the ability to carry cargo molecules across cell membranes. C-prune is the C-terminal domain of h-prune, overexpressed in breast, colorectal, and gastric cancers, interacting with multiple partners, and representing an ideal target for inhibition of cancer development. Its C-terminal domain results in an intrinsically disordered domain (IDD), and the peculiar properties of gH625 render it an optimal candidate to act as a carrier for this net negatively charged molecule by comparison with the positively charged TAT. A characterization of the recombinant gH625-c-prune fusion protein was conducted by biochemical, cellular biology and confocal microscopy means in comparison with TAT-c-prune. The results showed that the gH625-c-prune exhibited the ability to cross biomembranes, opening a new scenario on the use of gH625 as a novel multifunctional carrier.

A new cryptic cationic antimicrobial peptide from human apolipoprotein E with antibacterial activity and immunomodulatory effects on human cells

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.

Synthesis, spectroscopic studies and biological activity of a novel nucleopeptide with Moloney murine leukemia virus reverse transcriptase inhibitory activity.

In this work, we report the synthesis of an alternate nucleo-alpha,epsilon-peptide based on l-lysine moieties, an in vitro study of its biological activity, and spectroscopical binding studies between the novel nucleopeptide and Moloney murine leukemia virus reverse transcriptase as well as RNA. An alternate homothymine hexamer was synthesized by a straightforward solid phase route starting from commercial materials, purified by RP-HPLC and characterized by ESI-MS. The efficiency of the novel nucleo-alpha,epsilon-peptide in interfering with the reverse transcription of eukaryotic mRNA and the noteworthy enzymatic resistance demonstrated by specific assays are in favor of the employment of this nucleopeptide in novel biomedical strategies.

RGDechi-hCit: αvβ3 Selective Pro-Apoptotic Peptide as Potential Carrier for Drug Delivery into Melanoma Metastatic Cells

αvβ3 integrin is an important tumor marker widely expressed on the surface of cancer cells. Recently, we reported some biological features of RGDechi-hCit, an αvβ3 selective peptide antagonist. In the present work, we mainly investigated the pro-apoptotic activity of the molecule and its ability to penetrate the membrane of WM266 cells, human malignant melanoma cells expressing high levels of αvβ3 integrin. For the first time we demonstrated the pro-apoptotic effect and the ability of RGDechi-hCit to enter into cell overexpressing αvβ3 integrin mainly by clathrin- and caveolin-mediated endocytosis. Furthermore, we deepened and confirmed the selectivity, anti-adhesion, and anti-proliferative features of the peptide. Altogether these experiments give insight into the biological behavior of RGDechi-hCit and have important implications for the employment of the peptide as a new selective carrier to deliver drugs into the cell and as a therapeutic and diagnostic tool for metastatic melanoma. Moreover, since the peptide shows a pro-apoptotic effect, a great perspective could be the development of a new class of selective systems containing RGDechi-hCit and pro-apoptotic molecules or other therapeutic agents to attain a synergic action.

VEGFR1D2 in drug discovery: Expression and molecular characterization

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. Its biological activity is mediated by the binding to the extracellular domain of two tyrosine kinase transmembrane receptors: VEGFR1 and VEGFR2. Deletion studies showed that VEGF binding site resides in the first three domains of VEGFR1 and in domains 2 and 3 of VEGFR2. In particular, the second extracellular domain of VEGFR1 (VEGFR1(D2)) contains most of the VEGF binding requirements. Here, we report an efficient expression protocol and the molecular characterization by spectroscopic techniques of VEGFR1(D2). The protein was expressed in E. coli and refolded from inclusion bodies. The recombinant protein assumes the correct fold as assessed by a combination of biochemical and functional assays as well as by NMR characterization. Furthermore, the recombinant VEGFR1(D2) was analyzed by circular dichroism and fluorescence spectroscopy. The protein obtained by this procedure is suitable for the structural characterization of the complexes with receptor binders and to be used in interaction/screening studies.