Biomedical subjects

Lucia Falcigno

Publications and source records attributed to Lucia Falcigno.

7 recordsLinked to original sources

PubMed2006

Solution structure of mouse Cripto CFC domain and its inactive variant Trp107Ala.

We report here for the first time the solution structures at pH 3 and pH 6 of the synthetic CFC domain of mouse Cripto and of the point mutated variant W107A that is unable to bind to the Alk4 Cripto receptor. NMR data confirm that the CFC domain has a C1-C4, C2-C6, C3-C5 disulfide pattern and show that structures are rather flexible and globally extended, with three noncanonical antiparallel strands. His104 and Trp107 side chains protrude from a protein edge and are strongly exposed to solvent, supporting previous evidence of direct involvement in receptor binding. On the opposite molecule side, several nonpolar residues are gathered, forming a large hydrophobic patch that supposedly acts as interface with the cell membrane or the adjacent EGF-like domain. A second hydrophilic patch surrounding His104 and Trp107 is present only in the wild type variant, suggesting a possible involvement in modulating Alk4 recognition.

Amino Acid Sequence↗

PubMed2005

Effect of the addition of a nonionic surfactant on the complex poly(asparagine)--cationic surfactant.

Poly(asparagine) (pAsn) at 0.1wt % in the presence of dodecyltrimethylammonium bromide (DTAB) and pentaoxyethylene octyl ether (C(8)E(5)) at 1:1 molar ratio leads to the formation of mixed DTAB/C(8)E(5) micelle-like aggregates onto the polypeptide as a total surfactant critical association concentration (cac) is reached, as revealed by surface tension measurements and NMR chemical shifts. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) capable of revealing spatial relationships among proximal protons has been performed on the pAsn-DTAB-C(8)E(5)-water system to study structural details of the surfactant-polypeptide aggregates. NOESY cross-peaks at sample temperature of 298.15 K indicate that the polypeptide interacts with the DTAB/C(8)E(5) micelle-like aggregates. The NOE intermolecular effects also show direct interactions between surfactant and polypeptide in the pAsn-DTAB-water system, whereas no interaction has been revealed in the pAsn-C(8)E(5)-water system. Furthermore, the experimental evidence suggest that the DTAB-polypeptide complex is mainly driven by the polar attraction between the two molecules.

Cations↗

PubMed2005

Conformational analysis of heparin binding peptides.

A properly engineered biomaterial for dental/orthopaedic applications must induce specific responses from the osteoblasts at the implant site. A most desirable response is an efficient adhesion, as it represents the first phase in the cell/material interaction and the quality of this phase will influence the cell's capacity to organize into a new functional tissue. The four osteoblast-adhesive peptides discussed in this paper are mapped on the 339-364 sequence (339MAPRPSLAKKQRFRHRNRKGYRSQRG364) located in the primary heparin-binding site of human vitronectin (HVP). Adsorbed on a polystyrene scaffold, these peptides display different adhesive activities towards osteoblasts. In this paper we report on the structural analysis in solution of the peptides through NMR and computational techniques. We find that the peptides with the highest adhesive activities display a hydrophobic patch opposite to the charged surface candidate to interact with heparin. These findings suggest that the peptides might adsorb on the polystyrene support in a favourable orientation for their activity. Furthermore, molecular models obtained for the four peptides in solution were used in rigid docking simulations with a heparin model. Assuming that the peptide solution conformations are not very different from the polystyrene-adsorbed structures, the simulations reveal that peptide adhesive activity is also affected by the number of ionic interactions and spacing between charged residues.

Amino Acid Sequence↗

PubMed2004

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site 3: role of site-specific mutations.

Proteolytic processing of HIV gp160 to produce gp120 and gp41 is performed by PC enzymes. This process is a prerequisite for the virus infectivity, since both gp120 and gp41 participate in the virus HIV-1 entry mechanism. The structure of the gp120/gp41 junction remains to be elucidated, and the structural features required for molecular recognition between HIV-1 gp160 and proteolytic enzymes have not been clarified. Furin is the best PC candidate for the gp160 proteolytic processing known to date. In previous studies on model peptides, we have shown the relevance of an N-terminal helix for the proper recognition of the gp160 processing site by furin. Here we analyze the effect of point mutations in peptides lacking a regular N-terminal helix. To this end, we present the structure-activity characterization of three peptide analogues of the HIV gp160 processing site that all present mutations in proline at positions P3 and/or P2', while sharing the same N-terminal sequence, containing helix-breaking D-amino acids. Conformational analysis of the peptides was carried out in solution by NMR techniques, and furin's efficiency in cleaving them was measured. Structural findings are presented and discussed in relation to the different exhibited activity.

Catalytic Domain↗

PubMed2003

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site, 2: relevance of an N-terminal helix.

Proteolytic activation of the HIV-1 envelope glycoprotein gp160 is selectively performed by the proprotein convertase furin at the C-terminus of the sequence R508-E-K-R511 (site 1), in spite of the presence of another consensus sequence, Lys500-Ala-Lys-Arg503 (site 2). On the basis of the solution structural analysis of the synthetic peptide p498, spanning the gp160 sequence Pro498-Gly516, we previously suggested a possible role of an N-terminal helix in regulating the exposure and accessibility of the gp160 physiological cleavage site, enclosed in a loop. Here we report on the activity and conformation of the 23-residue peptide h-REKR, designed to exhibit a large N-terminal helix, followed by the gp160 native sequence, Arg508-Gly516. h-REKR is digested by furin with high efficiency, comparable to the full native p498. Circular dichroism analyses, in mixtures from pure water to 98 % trifluoroethanol, outline a significant content of helical structure in the peptide conformation. The molecular model obtained from NMR data collected in trifluoroethanol/water, by means of DYANA and AMBER simulations, indeed has helical structure on a large N-terminal segment. Such a long helix does not seem to affect the loop conformation of the C-terminal site 1-containing sequence, which exhibits the same proton chemical shifts already observed for the full native p498.

Amino Acid Sequence↗

PubMed2003

Hyaluronate tetrasaccharide- Cu(II) interaction: a NMR study.

The coordination of Cu(II) to a hyaluronate tetrasaccharide (HAt) was investigated in aqueous solution by 13C and 1H relaxation measurements at two magnetic fields, 9 and 14 T. The HAt interaction with the metal ion was monitored following the nuclear paramagnetic relaxation enhancements R1p and R2p produced by the copper addition. The data analysis shows that the paramagnetic effect is differently experienced by the nuclei in different monosaccharide residues. A molecular model for the complex HAt-Cu(II) was built taking into account the experimental data. The model shows the presence of two binding sites, both involving the carboxylate groups of the two glucuronic acid units. The first site, that best simulates the HA binding site, is located on the ligand core, while the second one is located on the terminal glucuronic acid residue. Both binding sites involve, in addition to the carboxylate groups, the O4 oxygens of the glucuronic acid residues.

Binding Sites↗

PubMed2002

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site.

The selective proteolytic activation of the HIV-1 envelope glycoprotein gp160 by furin and other precursor convertases (PCs) occurs at the carboxyl side of the sequence Arg508-Glu-Lys-Arg511 (site 1), in spite of the presence of another consensus sequence: Lys500-Ala-Lys-Arg503 (site 2). We report on the solution structural analysis of a 19-residue synthetic peptide, p498, which spans the two gp160-processing sites 1 and 2, and is properly digested by furin at site 1. A molecular model is obtained for p498, by means of molecular dynamics simulations, from NMR data collected in trifluoroethanol/water. The peptide N-terminal side presents a 9-residue helical segment, enclosing the processing site 2; the C-terminal segment can be described as a loop exposing the processing site 1. A hypothesis for the docking of p498 onto the catalytic domain of human furin, modeled by homology and fitting previous site-directed mutagenesis studies, is also presented. p498 site 1 is shown to have easy access to the furin catalytic site, unlike the nonphysiological site 2. Finally, on the basis of available data, we suggest a possible structural motif required for the gp160-PCs recognition.

Amino Acid Sequence↗