Biomedical subjects

Carlo Pedone

Publications and source records attributed to Carlo Pedone.

At least 19 recordsLinked to original sources

PubMed2007

Abeta(25-35) and its C- and/or N-blocked derivatives: copper driven structural features and neurotoxicity.

The toxic properties of beta-amyloid protein, Abeta(1-42), the major component of senile plaques in Alzheimer's disease, depend on nucleation-dependent oligomerization and aggregation. In addition, Abeta(1-42) toxicity is favored by the presence of trace metals, which affect the secondary structure of the peptide. A peptide comprising 11 residues within Abeta(1-42) [Abeta(25-35)] aggregates and retains the neurotoxic activity of Abeta(1-42). We have used both Abeta(25-35) and its C-amidated or N-acetylated/C-amidated derivatives to investigate the role of copper(II) in modulating the conformation and aggregation state as well as the neurotoxic properties of amyloid peptides. Electrospray ionization mass spectrometry (ESI-MS) and electron paramagnetic resonance (EPR) measurements were performed to verify the formation of copper(II)/Abeta(25-35) complexes and to determine the coordination mode, respectively. Abeta(25-35) and its derivatives were analyzed by circular dichroism spectroscopy to assess their secondary structure, subjected to thioflavine-T (Th-T) binding assay to reveal beta-sheet structured aggregates formation, and imaged by scanning force microscopy. Toxicity was assessed on mature cultures of rat cortical neurons. We found that beta-sheet-structured species of Abeta(25-35) were neurotoxic, whereas the random-coil-structured derivatives were devoid of effect. Interestingly, copper promoted the random-coil/beta-sheet transition of Abeta(25-35), with ensuing peptide toxicity, but it induced the toxicity of the N-acetylated/C-amidated derivative without affecting peptide folding. Moreover, copper did not influence either the folding or the activity of the C-amidated Abeta(25-35), suggesting that blockade of the C-terminus of Abeta peptides might be sufficient to prevent Abeta toxicity.

Amyloid beta-Peptides↗

PubMed2007

Does tetracycline bind helix 2 of prion? An integrated spectroscopical and computational study of the interaction between the antibiotic and alpha helix 2 human prion protein fragments.

We demonstrate here that tetracycline (TC) can strongly interact (KD' = 189 +/- 7 nM) with model peptides derived from the C-terminal globular domain of the prion protein, hPrP [173-195], and that interaction concerns residues within the C-terminal half of the helix 2, a short region previously indicated as endowed with ambivalent conformational behavior and implicated in PrP conversion to the beta-sheet-rich, infective scrapie variant. Data have been confirmed by binding studies with the N-terminal truncated 180-195 variant that displays a dissociation constant of 483 +/- 30 nM. Remarkably, TC does not influence the structure of the N-terminally fluoresceinated peptides that both show alpha-helical conformations. Docking calculations and molecular dynamics simulations suggest a direct, strong interaction of the antibiotic with exposed side chain functional groups of threonines 190-193 on the solvent-exposed surface of helix 2.

Amino Acid Sequence↗

PubMed2007

Nanostructures by self-assembling peptide amphiphile as potential selective drug carriers.

The self-assembling behavior, at physiological pH, of the amphiphile peptide (C18)(2)L5CCK8 in nanostructures is reported. Stable aggregates presenting a critical micellar concentration of 2 x 10(-6) mol kg(-1), and characterized by water exposed CCK8 peptide in beta-sheet conformation, are obtained. Small angle neutron scattering experiments are indicative for a 3D structure with dimensions > or =100 nm. AFM images confirm the presence of nanostructures. Fluorescence experiments indicating the sequestration of pyrene, chosen as drug model, and the anticancer Doxorubicin within the nanostructures are reported.

Antineoplastic Agents↗

PubMed2006

The chemical synthesis of the GstI protein by NCL on a X-Met site.

The small GstI protein (63 amino acids) of Rhizobium leguminosarum is the endogenous inhibitor of the glnII (glutamine synthetase II) gene expression. It has been suggested that GstI has a predominantly beta-structure and mediates the block of translation and stabilization of glnII mRNA through direct binding to its 5' untranslated region. Because of the unavailability of adequate amounts of purified recombinant protein, the mechanism as well as the protein tridimensional structure remain very poorly understood. To obtain the full-length protein, we have undertaken the chemical synthesis of the protein by different approaches. In a first attempt, the stepwise synthesis was unsuccessful, with strong aggregation experienced on the N-terminal side, after residue 44 from the C-terminus. In a second approach, we set up the conditions to carry out a native chemical ligation (NCL). Albeit the protein contains two Cysteine residues, located at positions 40 and 47, to minimize the size of the N-terminal segment to be synthesized, we have devised an alternative strategy of ligation on Met32, utilizing homoCys as the ligating moiety and then alkylating the resulting polypeptide with methyl iodide. New conditions to quantitatively methylate thiol groups in complex polypeptides have been conceived, obtaining the protein in very good yields and purity. A CD spectroscopy investigation has revealed that the protein does not adopt canonical secondary structures but is very rich in beta-structure (approximately 60%), in agreement with a previous study carried out on samples obtained by recombinant methods.

Amino Acid Sequence↗

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↗

PubMed2006

Antiproliferative activity of Pt(II) and Pd(II) phosphine complexes with thymine and thymidine.

Oxidative addition reactions between [M(PPh(3))(4)] (M=Pt and Pd) and N1-methylthymine (t)/3',5'-di-O-acetylthymidine (T) were carried out to give [M(II)(PPh(3))(2)Cl t (or T)] complexes, in which the metal is coordinated to the N3 of the base. All complexes were characterized by spectroscopic analyses (IR, NMR) and Fast Atom Bombardment mass spectrometry (FAB-MS); X-ray data for the thymine complexes and elemental analysis for the thymidine complexes are reported. The antiproliferative activity of the complexes was tested on human chronic myelogenous leukaemia K562 cells. Arrested polymerase-chain reaction analysis was carried on to correlate antiproliferative activity and inhibition of DNA replication. All Pd and Pt complexes exhibit antiproliferative activity, Pd complexes resulting always more active than Pt complexes. Arrested PCR data are strongly in agreement with the effects on cell growth, suggesting that inhibition of the DNA replication by the synthesized compounds is the major basis for their in vitro antiproliferative activity.

Base Sequence↗

PubMed2006

Carbonic anhydrase inhibitors: Hypoxia-activatable sulfonamides incorporating disulfide bonds that target the tumor-associated isoform IX.

An approach for designing bioreductive, hypoxia-activatable carbonic anhydrase (CA, EC 4.2.1.1) inhibitors targeting the tumor-associated isoforms is reported. Sulfonamides incorporating 3,3'-dithiodipropionamide/2,2'-dithiodibenzamido moieties were prepared and reduced enzymatically/chemically in conditions present in hypoxic tumors, leading to thiols. The X-ray crystal structure of the most promising compound, 4-(2-mercaptophenylcarboxamido)benzenesulfonamide, which as disulfide showed a K(I) against hCA IX of 653 nM (in reduced form of 9.1 nM), in adduct with hCA II showed the inhibitor making favorable interactions with Gln92, Val121, Phe131, Leu198, Thr199, Thr200, Pro201, and Pro202, whereas the sulfamoyl moiety was coordinated to the Zn2+ ion. The same interactions were preserved in the adduct with hCA IX, but in addition, a hydrogen bond between the SH moiety of the inhibitor and the amide nitrogen of Gln67 was evidenced, which may explain the almost 2 times more effective inhibition of the tumor-associated isozyme over the cytosolic isoform.

Antigens, Neoplasm↗

PubMed2006

Chemical synthesis of mouse cripto CFC variants.

We report for the first time the chemical synthesis of refolded CFC domain of mouse Cripto (mCFC) and of two variants bearing mutations on residues W107 and H104 involved in Alk4 binding. The domains undergo spontaneous and quantitative refolding in about 4 h, yet with very different kinetics. Disulfide linkages have been assessed by enzyme digestion and mass spectrometry analysis of resulting fragments, and the first experimental studies on structural organization have been conducted by circular dichroism spectroscopy under different pH conditions. Upon refolding, the domains considerably change their conformations, although they do not assume canonical structures, and become highly resistant to enzyme degradation. A comparative study of receptor binding shows that the CFC domain can bind Alk4 and confirms the importance of W107 and H104 for receptor recognition.

Activin Receptors, Type I↗

PubMed2006

A novel member of the protein disulfide oxidoreductase family from Aeropyrum pernix K1: structure, function and electrostatics.

The formation of disulfide bonds between cysteine residues is a rate-limiting step in protein folding. To control this oxidative process, different organisms have developed different systems. In bacteria, disulfide bond formation is assisted by the Dsb protein family; in eukarya, disulfide bond formation and rearrangement are catalyzed by PDI. In thermophilic organisms, a potential key role in disulfide bond formation has recently been ascribed to a new cytosolic Protein Disulphide Oxidoreductase family whose members have a molecular mass of about 26 kDa and are characterized by two thioredoxin folds comprising a CXXC active site motif each. Here we report on the functional and structural characterization of ApPDO, a new member of this family, which was isolated from the archaeon Aeropyrum pernix K1. Functional studies have revealed that ApPDO can catalyze the reduction, oxidation and isomerization of disulfide bridges. Structural studies have shown that this protein has two CXXC active sites with fairly similar geometrical parameters typical of a stable conformation. Finally, a theoretical calculation of the cysteine pK(a) values has suggested that the two active sites have similar functional properties and each of them can impart activity to the enzyme. Our results are evidence of functional similarity between the members of the Protein Disulphide Oxidoreductase family and the eukaryotic enzyme PDI. However, as the different three-dimensional features of these two biological systems strongly suggest significantly different mechanisms of action, further experimental studies will be needed to make clear how different three-dimensional structures can result in systems with similar functional behavior.

Aeropyrum↗

PubMed2006

Molecular dynamics analyses of cross-beta-spine steric zipper models: beta-sheet twisting and aggregation.

The structural characterization of amyloid fibers is one of the most investigated areas in structural biology. The structural motif, denoted as steric zipper, recently discovered for the peptide GNNQQNY [Nelson, R., Sawaya, M. R., Balbirnie, M., Madsen, A. O., Riekel, C., Grothe, R. & Eisenberg, D. (2005) Nature 435, 773-778], is expected to exert strong influence in this field. To obtain further insights into the features of this unique structural motif, we report several molecular dynamics simulations of various GNNQQNY aggregates. Our analyses show that even pairs of beta-sheets composed of a limited number of beta-strands are stable in the 20-ns time interval considered, which suggests that steric zipper interactions at a beta-sheet-beta-sheet interface strongly contribute to the stability of these aggregates. Moreover, although the basic features of side chain-side chain interactions are preserved in the simulation, the backbone structure undergoes significant variations. Upon equilibration, a significant twist of the beta-strands that compose the beta-sheets is observed. These results demonstrate that the occurrence of steric zipper interactions is compatible with flat and twisted beta-sheets. Molecular dynamics simulations carried out on two pairs of beta-sheets, separated in the crystal state by a hydrated interface, lead to interesting results. The two pairs of sheets, while twisting, associate through stable peptide-peptide interactions. These findings provide insight into the mechanism that leads to the formation of higher aggregates. On these bases, it is possible to reconcile the crystallographic and the EM data on the size of the basic GNNQQNY fiber unit.

Amino Acid Sequence↗

PubMed2006

Carbonic anhydrase inhibitors: X-ray and molecular modeling study for the interaction of a fluorescent antitumor sulfonamide with isozyme II and IX.

The X-ray crystal structure of the fluorescent antitumor sulfonamide carbonic anhydrase (CA, EC, 4.2.1.1) inhibitor (4-sulfamoylphenylethyl)thioureido fluorescein (1) in complex with the cytosolic isoform hCA II is reported, together with a modeling study of the adduct of 1 with the tumor-associated isoform hCA IX. Its binding to hCA II is similar to that of other benzesulfonamides, with the ionized sulfonamide coordinated to the Zn2+ ion within the enzyme active site, and also participating in a network of hydrogen bonds with residues Thr199 and Glu106. The scaffold of 1 did not establish polar interactions within the enzyme active site but made hydrophobic contacts (<4.5 A) with Gln92, Val121, Phe131, Val135, Leu198, Thr199, Thr200, and Pro202. The substituted 3-carboxy-amino-phenyl functionality was at van der Waals distance from Phe131, Gly132, and Val135. The bulky tricyclic fluorescein moiety was located at the rim of the active site, on the protein surface, and strongly interacted with the alpha-helix formed by residues Asp130-Val135. All these interactions were preserved in the hCA IX-1 adduct, but the carbonyl moiety of the fluorescein tail of 1 participates in a strong hydrogen bond with the guanidine moiety of Arg130, an amino acid characteristic of the hCA IX active site. This may account for the roughly 2 times higher affinity of 1 for hCA IX over hCA II and may explain why in vivo the compound specifically accumulates only in hypoxic tumors overexpressing CA IX and not in the normal tissues. The compound is in clinical studies as an imaging tool for acute hypoxic tumors.

Amino Acid Sequence↗

PubMed2006

Novel and selective alpha(v)beta3 receptor peptide antagonist: design, synthesis, and biological behavior.

Among RGD-dependent integrins, the alpha(v)beta3 receptor has recently received increasing attention as a therapeutic target because of its critical role in tumor-induced angiogenesis and metastasis formation. Here, we describe a new peptide antagonist of alpha(v)beta3 receptor, designed on the basis of the crystal structure of integrin alpha(v)beta3 in complex with c(RGDf[NMe]V) and the NMR structure of echistatin. Cell adhesion assays have demonstrated that the peptide is a potent and selective antagonist of the alpha(v)beta3 receptor.

Cell Adhesion↗

PubMed2006

Structural requirements for proinflammatory activity of porin P2 Loop 7 from Haemophilus influenzae.

Haemophilus influenzae type b (Hib) is one of the leading causes of invasive bacterial infection in young children, characterized by inflammation mainly mediated by cytokines and chemokines. One of the most abundant components of the Hib outer membrane is the P2 porin, which has been shown to induce the release of several inflammatory cytokines. Synthetic peptides corresponding to loops L5, L6, and L7 activate JNK and p38 mitogen-activated protein kinase (MAPK) pathways, L7 being the most active peptide. Therefore, sequence-activity relationships and key residues were identified by elongating sequence to different extents, designing cyclic peptides, and performing an alanine scan of L7. The ability of mutant peptides to induce activation of signal transduction pathways and release of TNF-alpha and IL-6 has been determined, and, in conjunction with CD spectra, bioinformatics analysis, and molecular dynamics data, showed that 6 out of 8 amino acids contribute significantly to the overall activity. Molecular dynamics showed that L7 modifications increased loop rigidity and helicity after Gly6 mutation, thus, providing a possible structural explanation for observed loss of bioactivity. This work provides insights into essential molecular details of P2 that may impact on the pathogenesis of Hib infections where interruption of the signaling cascade could represent an attractive therapeutic strategy.

Amino Acid Motifs↗

PubMed2006

Structure/function of KRAB repression domains: structural properties of KRAB modules inferred from hydrodynamic, circular dichroism, and FTIR spectroscopic analyses.

The abundant zinc finger proteins (ZFPs) sharing the KRAB motif, a potent transcription repression domain, direct the assembly on templates of multiprotein repression complexes. A pivotal step in this pathway is the assembly of a KRAB domain-directed complex with a primary corepressor, KAP1/KRIP-1/TIF1beta. The structure/function dependence of KRAB/TIF1beta protein-protein interaction and properties of the complex, therefore, play pivotal roles in diverse cellular processes depending on KRAB-ZFPs regulation. KRAB domains are functionally bipartite. The 42 amino acid-long KRAB-A module, indeed, is necessary and sufficient for transcriptional repression and for the interaction with the tripartite RBCC region of TIF1beta, while the KRAB-B motif seems to potentiate the assembly of the complex. The structural properties of KRAB-A and KRAB-AB domains from the human ZNF2 protein have been investigated by characterizing highly purified lone (A) and composite (AB) modules. Hydrodynamic and spectroscopic features, investigated by means of gel filtration, circular dichroism, and infrared spectroscopy, provide evidence that both KRAB-A and KRAB-AB domains present low compactness, structural disorder, residual secondary structure content, flexibility, and tendency to molecular aggregation. Comparative analysis among KRAB-A and KRAB-AB modules suggests that the presence of the -B module may influence the properties of lone KRAB-A by affecting the structural flexibility and stability of the conformers. The combined experimental data and the intrinsic features of KRAB-A and KRAB-AB primary structures indicate a potential role of specific subregions within the modules in driving structural flexibility, which is proposed to be of importance for their function.

Amino Acid Sequence↗

PubMed2006

Metal ion substitution in the catalytic site greatly affects the binding of sulfhydryl-containing compounds to leucyl aminopeptidase.

Bovine lens leucyl aminopeptidase (blLAP), a homohexameric metallopeptidase preferring bulky and hydrophobic amino acids at the N-terminus of (di)peptides, contains two Zn(2+) ions per subunit that are essential for catalytic activity. They may be replaced by other divalent cations with different exchange kinetics. The protein readily exchangeable site (site 1) can be occupied by Zn(2+), Mn(2+), Mg(2+), or Co(2+), while the tight binding site (site 2) can be occupied by Zn(2+) or Co(2+). We recently reported that introduction of Mn(2+) into site 1 generates a novel activity of blLAP toward CysGly [Cappiello, M., et al. (2004) Biochem. J. 378, 35-44], which in contrast is not hydrolyzed by the (Zn/Zn) enzyme. This finding, while disclosing a potential specific role for blLAP in glutathione metabolism, raised a question about the features required for molecules to be a substrate for the enzyme. To clarify the interaction of the enzyme with sulfhydryl-containing derivatives, (Zn/Zn)- and (Mn/Zn)blLAP forms were prepared and functional-structural studies were undertaken. Thus, a kinetic analysis of various compounds with both enzyme forms was performed; the crystal structure of (Zn/Zn)blLAP in complex with the peptidomimetic derivative Zofenoprilat was determined, and a modeling study on the CysGly-(Zn/Zn)blLAP complex was carried out. This combined approach provided insight into the interaction of blLAP with sulfhydryl-containing derivatives, showing that the metal exchange in site 1 modulates binding to these molecules that may result in enzyme substrates or inhibitors, depending on the nature of the metal.

Binding Sites↗

PubMed2006

Conformationally constrained CCK8 analogues obtained from a rationally designed peptide library as ligands for cholecystokinin type B receptor.

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.

Cholecystokinin↗

PubMed2006

A short PNA targeting coxsackievirus B3 5'-nontranslated region prevents virus-induced cytolysis.

Targeting regulatory RNA regions to interfere with the biosynthesis of a protein is an intriguing alternative to targeting a protein itself. Regulatory regions are often unique in sequence and/or structure and, thus, ideally suited for specific recognition with a low risk of undesired side effects. Targeting regulatory RNA elements, however, is complicated by their complex three-dimensional structure, which poses kinetic and thermodynamic constraints to the recognition by a complementary oligonucleotide. Oligonucleotide mimics, which shift the thermodynamic equilibrium towards complex formation and yield stable complexes with a target RNA, can overcome this problem. Peptide nucleic acids (PNA) represent such a promising class of molecules. PNA are very stable, non-ionic compounds and they are not sensitive to enzymatic degradation. Yet, PNA form specific base pairs with a target sequence. We have designed, synthesised and characterised PNA able to enter infected cells and to bind specifically to a control region of the genomic RNA of coxsackievirus B3 (CVB3), which is an important human pathogen. The results obtained by studying the interaction of such PNA with their RNA target, the entrance into the cell and the viral inhibition are herein presented.

Antiviral Agents↗

PubMed2006

Analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type 1 glycoproteins H and B.

Human herpesviruses enter cells by fusion of their own membrane with a cellular membrane through the concerted action of multiple viral proteins and cellular receptors. Two conserved viral glycoproteins, gB and gH, are required for herpes simplex virus type 1 (HSV-1)-mediated membrane fusion, but little is known of how these proteins cooperate during entry. Both glycoproteins were shown to contain heptad repeat (HR) sequences predicted to form alpha-helical coiled coils, and the inhibitory activity against infection of four sets of synthetic peptides corresponding to HR1 and HR2 of gB and gH was tested. The interactions between these HR peptides were also investigated by circular dichroism, native polyacrylamide-gel electrophoresis and size exclusion high-performance liquid chromatography. gH coiled-coil peptides were more effective than gB coiled-coils peptides in inhibiting virus infectivity. The peptides did not impair fusion when added to cells immediately after infection. In contrast, inhibition of infection was observed, albeit to various extents, when peptides were added to virus before or during inoculation. The results of biophysical analyses were indicative of the existence of an interaction between HR1 and HR2 of gH and suggest that the HRs of gB and gH do not interact with each other.

Amino Acid Sequence↗