Antonio Palleschi | Università di Roma Tor Vergata (original) (raw)

Papers by Antonio Palleschi

Research paper thumbnail of Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis

Human Molecular Genetics, Apr 4, 2014

RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dys... more RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits and predisposition to certain malignancies, are caused by constitutional dysregulation of RAS signalling predominantly through the RAF/MEK/ERK (MAPK) cascade. We report on two germline mutations (p.Gly39dup and p.Val55Met) in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare (2 subjects among 504 individuals analysed) and variable phenotype with features partially overlapping Noonan syndrome, the most common RASopathy. We also identified somatic RRAS mutations (p.Gly39dup and p.Gln87Leu) in 2 of 110 cases of non-syndromic juvenile myelomonocytic leukaemia, a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signalling, defining an atypical form of this haematological disorder rapidly progressing to acute myeloid leukaemia. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in Caenorhabditis elegans enhanced RAS signalling and engendered protruding vulva, a phenotype previously linked to the RASopathy-causing SHOC2 S2G mutant. Overall, these findings provide evidence of a functional link between RRAS and MAPK signalling and reveal an unpredicted role of enhanced RRAS function in human disease.

Research paper thumbnail of A Nitroxide Derivative as a Probe for Conformational Studies of Short Linear Peptides in Solution. Spectroscopic and Molecular Mechanics Investigation

Journal of Physical Chemistry B, Sep 15, 1998

The photophysics of linear Aib-based hexapeptides (Aib) R-aminoisobutyric acid) of general formul... more The photophysics of linear Aib-based hexapeptides (Aib) R-aminoisobutyric acid) of general formula Ac-Toac-(Aib) n-Trp-(Aib) r-OtBu (TnTrp), where n + r) 4, and Toac and Trp are a nitroxide spin labeled C R,R-disubstituted glycine and tryptophan, respectively, were investigated in methanol and dioxane solutions by steady-state and time-resolved fluorescence measurements. Another hexapeptide, i.e., Boc-(S)Bin-Ala-Aib-Toac-(Ala) 2-OtBu (T-Bin), carrying the same nitroxide derivative and a binaphthyl as the fluorophore was also studied by the same techniques. Quenching of the excited tryptophan or binaphthyl chromophore proceeds on a time scale from subnanoseconds to a few nanoseconds, depending on the conformers distribution in solution. CD and IR spectral patterns in methanol or CDCl 3 suggest that the backbone of the peptides examined is in the 3 10-helical conformation, thus preserving the structural features of the crystal state, as earlier determined by X-ray diffraction measurements. The fluorescence results were satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited tryptophan or binaphthyl to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process. Molecular mechanics calculations coupled with time decay data allowed us to build up the most probable structures of these peptides in methanol solution.

Research paper thumbnail of Carnitine palmitoyl transferase-1A (CPT1A): a new tumor specific target in human breast cancer

Oncotarget, Jan 21, 2016

Transcriptional mechanisms epigenetically-regulated in tumoral tissues point out new targets for ... more Transcriptional mechanisms epigenetically-regulated in tumoral tissues point out new targets for anti-cancer therapies. Carnitine palmitoyl transferase I (CPT1) is the rate-limiting enzyme in the transport of long-chain fatty acids for β-oxidation. Here we identified the tumor specific nuclear CPT1A as a product of the transcript variant 2, that doesn't retain the classical transferase activity and is strongly involved in the epigenetic regulation of cancer pro-survival, cell death escaping and tumor invasion pathways. The knockdown of CPT1A variant 2 by small interfering RNAs (siRNAs), was sufficient to induce apoptosis in MCF-7, SK-BR3 and MDA-MB-231 breast cancer cells. The cell death triggered by CPT1A silencing correlated with reduction of HDAC activity and histone hyperacetylation. Docking experiments and molecular dynamics simulations confirmed an high binding affinity of the variant 2 for HDAC1. The CPT1A silenced cells showed an up-regulated transcription of pro-apoptotic genes (BAD, CASP9, COL18A1) and down-modulation of invasion and metastasis related-genes (TIMP-1, PDGF-A, SERPINB2). These findings provide evidence of the CPT1 variant 2 involvement in breast cancer survival, cell death escape and invasion. Thus, we propose nuclear CPT1A as a striking tumor specific target for anticancer therapeutics, more selective and effective as compared with the well-known HDAC inhibitors.

Research paper thumbnail of Ultrasound-Stimulated PVA Microbubbles as a Green and Handy Tool for the Cleaning of Cellulose-Based Materials

Gels, Jun 23, 2023

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Polymer-supported catalysis: stereoselective oxidation of L-dopa by iron(III) complex ions anchored to asymmetric polymers

Macromolecules, Mar 1, 1986

ABSTRACT

Research paper thumbnail of A selective chemoreceptor for Aflatoxin B1

Aflatoxin B1 (AFB1) is a toxin produced by the mould fungus varieties Aspergillus Flavus and Aspe... more Aflatoxin B1 (AFB1) is a toxin produced by the mould fungus varieties Aspergillus Flavus and Aspergillus parasiticus, which grows on foodstuffs like peanuts, flour, pepper etc. Due to its high toxicity for the human health, the AFB1 allowed concentration in food and animal feed is limited to ppb according to last EC directives [1]. At present, the methods available for aflatoxin detection are based mainly on HPLC or ELISA techniques. Therefore, new methods with low detection limit and sufficient selectivity towards AFB1 with respect to its less toxic analogous molecules, aflatoxins G1 (AFG1) and G2 (AFG2), need to be developed. Furthermore, portable and simple methods are preferred for the possibility to be used directly on site. In this respect, by using computer modelling, we have identified a chemoreceptor, based on a porphyrin moiety, able to bind selectively AFB1 with respect to AFG1. These predictions were confirmed by evaluating the binding properties of different porphyrin molecules with AFB1, AFG1 and AFG2 in THF. The system was fully characterised by means of UV absorption and steady-state and time resolved fluorescence measurements.

Research paper thumbnail of New Strategy for the Cleaning of Paper Artworks: A Smart Combination of Gels and Biosensors

Advances in chemistry, Sep 2, 2014

In this work an outlook on the design and application, in the cultural heritage field, of new too... more In this work an outlook on the design and application, in the cultural heritage field, of new tools for diagnostic and cleaning use, based on biocompatible hydrogels and electrochemical sensors, is reported. The use of hydrogels is intriguing because it does not require liquid treatment that could induce damage on artworks, while electrochemical biosensors not only are easy to prepare, but also can be selective for a specific compound and therefore are suitable for monitoring the cleaning process. In the field of restoration of paper artworks, more efforts have to be done in order to know how to perform the best way for an effective restoration. Rigid Gellan gel, made up of Gellan gum and calcium acetate, was proposed as a paper cleaning treatment, and selective biosensors for substances to be removed from this gel have been obtained by choosing the appropriate enzymes to be immobilized. Using this approach, it is possible to know when the cleanup process will be completed, avoiding lengthy and sometimes unnecessary cleaning material applications.

Research paper thumbnail of Germline Missense Mutations Affecting KRAS Isoform B Are Associated with a Severe Noonan Syndrome Phenotype

American Journal of Human Genetics, Jul 1, 2006

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphi... more Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring-binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor.

Research paper thumbnail of Conformational calculations restrained by NMR and fluorescence data on functionalized Aib-based peptides

Kluwer Academic Publishers eBooks, Dec 2, 2005

ABSTRACT

Research paper thumbnail of A New Concept: A Peptide Monolayer for Accurate Positioning of Electroactive Probes at Close Distance from a Gold Surface

Research paper thumbnail of Peptide-sandwiched protoporphyrin compounds mimicking hemoprotein structures in solution

Biopolymers, 2000

A series of covalently bound peptide-protoporphyrin-peptide compounds, also carrying naphthalene ... more A series of covalently bound peptide-protoporphyrin-peptide compounds, also carrying naphthalene (N) to allow a photophysical investigation, were synthesized. Their general formula is P(nN)(2), where P refers to protoporphyrin IX, and n to the number of amino acids in the sequence Boc-Leu-Leu-Lys-(Ala)(x) -Leu-Leu-Lys-OtBu of each backbone chain (x = 0-3; n = x + 6). Their structural features in methanol solution were investigated by ir and CD spectra, and by steady-state and time resolved fluorescence experiments as well. The ir spectra indicate that intramolecularly H-bonded conformations form, and CD data in both methanol and water-methanol mixture suggest the presence of alpha-helix structure. Quenching of excited naphthalene takes place by electronic energy transfer from singlet N* to P ground state. Fluorescence decays coupled with molecular mechanics calculations indicate that two conformers for each dimeric peptide are the major contributors to the observed phenomena. These conformers are characterized by a globular, protein-like structure, where the protoporphyrin resides in a central pocket, while the two N groups are externally situated. Of the four N linkages in the two conformers, three of them attain a very similar steric arrangement around the central P molecule, in terms of both center-to-center distance and mutual orientation, while the fourth experiences a different steric disposition as compared to the others. Experimental photophysical parameters satisfactorily compare with those obtained by theoretical calculations, within the Förster mechanism for long-range energy transfer, only when the mutual orientation of the chromophores was also taken into account. This implies that interconversion among conformational substates of probes linkages is slow on the time scale of the energy transfer process.

Research paper thumbnail of Controlling the Formation of Peptide Films: Fully Developed Helical Peptides are Required to Obtain a Homogenous Coating over a Large Area

Collection of Czechoslovak Chemical Communications, Nov 1, 2019

The influence of conformational dynamics on the self-assembly process of a novel, conformationall... more The influence of conformational dynamics on the self-assembly process of a novel, conformationally constrained, analogue of the natural, antimicrobial peptide trichogin GA IV was analysed by spectroscopic methods, microscopy imaging at nanometric resolution, and molecular dynamics simulations. The formation of antimicrobial peptide films at the air/water interface and their deposition on a graphite or a mica substrate were investigated. Combining experimental evidence with molecular dynamics simulation, we demonstrate that only the fully-developed helical structure of the analogue promotes formation of ordered aggregates nucleating the growth of micrometric rods, that give rise to homogenous coating over wide regions of the hydrophilic mica. This work proves the influence of helix flexibility on peptide self-organization and orientation on surfaces, key steps in the interaction of antimicrobial peptides with their target biological membranes.

Research paper thumbnail of Photophysical Properties of 1,3,5-Tris(2-naphthyl)benzene and Related Less-Arylated Compounds: Experimental and Theoretical Investigations

Journal of Physical Chemistry A, Aug 14, 2009

Recently, a growing interest has concerned compounds characterized by high chemical and photophys... more Recently, a growing interest has concerned compounds characterized by high chemical and photophysical stability and high quantum yield for their possible technological applications. 1,3,5-Tris(2-naphthyl)benzene (N3B), 1,3-bis(2-naphthyl)benzene (N2B), and 2-naphthyl-benzene (N1B) are promising compounds, but they needed a detailed photophysical characterization. In this context, theoretical and experimental investigations have been carried out. Steady-state and decay time fluorescence measurements indicate that the second naphthyl group, added in the meta position of N1B, perturbs the electronic levels, whereas the further naphthyl addition, leading to N3B, does not promote changes in all of the observed properties. The investigated compounds show a biexponential fluorescence decay that has been attributed to a rearrangement involving the exited states S(1) and S(2). The minimum structure corresponding to the S(1) and S(2) states has been obtained at the configuration interaction with single excitations (CIS) level of theory. For the ground-state geometry, a conformational analysis at the Hartree-Fock level has also been carried out. We have evaluated the energy gaps between electronic levels by using Zerner's intermediate neglect of differential overlap (ZINDO) method. The species involved in the fluorescence have been experimentally characterized, and the decay-associated spectra have been obtained.

Research paper thumbnail of A spectroscopic and molecular mechanics investigation on a series of AIB-based linear peptides and a peptide template, both containing tryptophan and a nitroxide derivative as probes

Biopolymers, Feb 1, 2000

Linear Aib-based hexapeptides, of the general formula Ac-Toac-(Aib)(n) -Trp-(Aib)(r) -OtBu [T(Aib... more Linear Aib-based hexapeptides, of the general formula Ac-Toac-(Aib)(n) -Trp-(Aib)(r) -OtBu [T(Aib)(n) Trp], where n + r = 4, and Toac is a nitroxide spin-labeled C(alpha,alpha)-disubstituted glycine, were investigated by steady-state and time-resolved fluorescence measurements in different solvent media. A related peptide, i.e., cyclo-¿Orn-[(Aib)(2)-Trp-(Aib)(2)-Z]-Asp-[(Aib)(2)-Toac-(Aib)(2)-+ ++OtBu ]¿ [T-cyclo-Trp], was also studied by the same techniques. It is a L-Orn, L-Asp diketopiperazine template, to which two Aib-based chains are covalently attached, each one containing one chromophore only, i.e., Trp or Toac. Whatever the solvent, in the former series of peptides quenching of the excited Trp exhibits three lifetime components and proceeds on a time scale from subnanoseconds to a few nanoseconds, while in the case of the template the same process occurs entirely on the nanoscale time scale, exhibiting two lifetimes only. The ir absorption spectral patterns suggest that the backbone of the peptides examined is in the 3(10)-helical conformation, as earlier determined by x-ray diffraction for T(Aib)(3)Trp in the crystal state. In all cases, the fluorescence results are satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited Trp to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process, allowing us to estimate the dynamics of the process. Molecular mechanics calculations coupled with time decay data made it possible to build up the most probable structures of these peptides in solution.

Research paper thumbnail of Interdisciplinary approach to develop a disposable real time monitoring tool for the cleaning of graphic artworks. Application on “le Nozze di Psiche”

Microchemical Journal, May 1, 2018

Disposable non-invasive and compatible real time monitoring tool was developed in order to follow... more Disposable non-invasive and compatible real time monitoring tool was developed in order to follow the cleaning process of paper artwork directly in situ. This tool was based on a biocompatible cleaning hydrogel coupled with flow electrochemical diagnostic tool, suitable to verify in situ and in a simple way the assessment of degradation of artwork and the efficiency of cleaning process. In this paper, the results obtained by applying this tool on a great format artwork with a lining as support, "Le Nozze di Psiche", engraved by Diana Scultori, printed in 1613, are reported. This opera was affected by a structural and chromatic deterioration due to a strong oxidative degradation. Such deterioration was probably accelerated by the adhesive (a mixture of starch paste and animal glue) used in a previous lining intervention. In this case, the cleaning agents used are rigid hydrogels of Gellan gum, modified with hydrolytic enzymes. By using the flow sampling system, all materials removed by the gel was carried up to a thin layer cell containing a selective electrochemical biosensors, suitable to monitor both treatments, the cleaning process and the removal of lining. These were monitored, allowing understanding when both processes were completed, thus avoiding lengthy and unnecessary cleaning applications. The effectiveness of cleaning with Gellan gel was assessed quantitatively by using non-invasive optical reflectance spectroscopy by a portable instrumentation, elaborating data with an improved version of the Kubelka-Munk theory in order to recover the absorption coefficient of the cellulose fibers of "Le Nozze di Psiche". The concentration of oxidized groups acting as chromophores was obtained by comparing the experimental optical absorption spectra to those simulated computationally by using TDDFT-based calculations. By following the cleaning with Gellan gel the results indicate a large decrease of the concentration of degradation product of cellulose. Moreover, chromatographic analysis were carried out in order to evaluate the amount of acid compound, produced during the aging and present on the graphic artwork, using the Gellan gel after cleaning step. The results obtained from the restoration of "Le Nozze di Psiche" have allowed the restorers to evaluate innovative methods for cleaning treatment of paper artworks with a highly specialized scientific-diagnostic approach.

Research paper thumbnail of Molecular dynamics methods to predict peptide locations in membranes: LAH4 as a stringent test case

Biochimica Et Biophysica Acta - Biomembranes, Feb 1, 2015

Determining the structure of membrane-active peptides inside lipid bilayers is essential to under... more Determining the structure of membrane-active peptides inside lipid bilayers is essential to understand their mechanism of action. Molecular dynamics simulations can easily provide atomistic details, but need experimental validation. We assessed the reliability of self-assembling (or "minimum-bias") and potential of mean force (PMF) approaches, using all-atom (AA) and coarse-grained (CG) force-fields. The LAH4 peptide was selected as a stringent test case, since it is known to attain different orientations depending on the protonation state of its four histidine residues. In all simulations the histidine side-chains inserted in the membrane when neutral, while they interacted with phospholipid headgroups in their charged state. This led to transmembrane orientations for neutral-His LAH4 in all minimum-bias AA simulations and in most CG trajectories. By contrast, the charged-His peptide stabilized membrane defects in AA simulations, whereas it was located at the membrane surface in some CG trajectories, and interacted with both lipid leaflets in others. This behavior is consistent with the higher antimicrobial activity and membrane-permeabilizing behavior of the charged-His LAH4. In addition, good agreement with solid-state NMR orientational data was observed in AA simulations. PMF calculations correctly predicted a higher membrane affinity for the neutral-His peptide. Interestingly, the structures and relative populations of PMF local free-energy minima corresponded to those determined in the less computationally demanding minimum-bias simulations. These data provide an indication about the possible membrane-perturbation mechanism of the charged-His LAH4 peptide: by interacting with lipid headgroups of both leaflets through its cationic side-chains, it could favor membrane defects and facilitate translocation across the bilayer.

Research paper thumbnail of The importance of being kinked: role of Pro residues in the selectivity of the helical antimicrobial peptide P5

Journal of Peptide Science, Oct 31, 2013

Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against... more Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against drug-resistant bacteria. Many of them kill bacteria by perturbing their membranes but exhibit no significant toxicity towards eukaryotic cells. The identification of the features responsible for this selectivity is essential for their pharmacological development. AMPs exhibit few conserved features, but a statistical analysis of an AMP sequence database indicated that many α-helical AMPs surprisingly have a helix-breaking Pro residue in the middle of their sequence. To discriminate among the different possible hypotheses for the functional role of this feature, we designed an analogue of the antimicrobial peptide P5, in which the central Pro was deleted (analogue P5Del). Pro removal resulted in a dramatic increase of toxicity. This was explained by the observation that P5Del binds both charged and neutral membranes, whereas P5 has no appreciable affinity towards neutral bilayers. CD and simulative data provided a rationalization of this behavior. In solution P5, due to the presence of Pro, attains compact conformations, in which its apolar residues are partially shielded from the solvent, whereas P5Del is more helical. These structural differences reduce the hydrophobic driving force for association of P5 to neutral membranes, whereas its binding to anionic bilayers can still take place because of electrostatic attraction. After membrane binding, the Pro residue does not preclude the attainment of a membrane-active amphiphilic helical conformation. These findings shed light on the role of Pro residues in the selectivity of AMPs and provide hints for the design of new, highly selective compounds.

Research paper thumbnail of Chiroptical, IR And Fluorescence Spectra of Short Linear Peptides in Methanol and Water-Methanol Mixtures

Springer eBooks, 1995

ABSTRACT

Research paper thumbnail of The Role of Thermodynamics in the Activity and Selectivity of Antimicrobial Peptides

Biophysical Journal, Feb 1, 2016

Research paper thumbnail of Electron transfer in catecholamines - tetrapyridine-iron(III) complex ions anchored to polypeptides

Inorganica Chimica Acta, 1983

Abstract [Fe(tetpy)(OH) 2 ] + complex ions (tetpy = 2,2′,2′',2‴-tetrapyridyl) anchored to pol... more Abstract [Fe(tetpy)(OH) 2 ] + complex ions (tetpy = 2,2′,2′',2‴-tetrapyridyl) anchored to poly(L-glutamate) (FeL) or poly(D-glutamate) (FeD) through the γ-carboxylate groups of the polymers [1] exhibit stereoselective peroxidatic activity with a number of optically active substrates, according to the reaction (pH = 7.0, Tris buffer 0.05 M ) [2, 3]: Progressive binding of complex ions was found to determine a coil-to-α-helix transition in the charged polypeptide matrices, as well as aggregation phenomena with a freezing of iron molecules inbetween helical chains. Under these conditions, electron transfer from catecholamines (L-adrenalin and L-dopa) to the central iron(III) ion, which is rate-determining, proceeds stereoselectively because extensive and possibly specific interactions between substrate molecules and the peptidic residues in the close environment of the active sites different steric constraints for the two diastereomeric precursor complexes. The rate constants of electron transfer processes can be expressed by nuclear and electronic factors which are highly sensitive to the separation of the redox centers [4]. Therefore, even small differences in steric hindrances between the diastereo-isomers would affect differently the optimal mutual orientation of the reacting OH group of the substrates and the peripheral tetrapyridyl ligand of the active sites, whose π-system very likely acts as an electron-transfer agent [5]. Preliminary results on conformational analysis of substrate-catalyst adducts support such conclusions. This is reflected in the rate constants of the electron-transfer step, as illustrated by the Lineweaver-Burk plot of the reaction catalyzed by FeL or FeD systems at a complex to polymer-residue ratio [C]/[P] = 0.20, reported in Fig. 1. From the results, the turnover numbers for the oxidation of L-adrenaline are 0.56±0.07 and 2.04±0.23 min −1 , respectively, whereas those for the oxidation of L-dopa are 0.42±0.05 and 1.14±0.12 min −1 (25.9 °C). On the other hand, the Michaelis constants are K M = (1.30±0.18) × 10 −3 and (1.05±0.15) × 10 −3 M in the former case, and (8.0±1.12) × 10 −4 and (5.50±0.48) × 10 −4 M in the latter. According to Marcus theory [6], the ultimate rate of electron transfer depends on the rate of reorganization of the surrounding medium (precursor and successor complex formation). When the asymmetric polymers play an active role in the reaction, being involved in the formation of the precursor complex the polypeptide itself might experience local conformational changes. Since the time scale of these rearrangements is unknown, it is possible that the polymer reorganization retards the overall rate of the electron transfer process. This could explain the finding that stereoselectivity occurs at the expense of the efficiency of reaction, as shown in Fig. 2. Using catalysts at low [C]/[P] ratio, the active sites are exposed to the bulk solvent and a substrate-coordinated metal chelate forms without any assistance from the coiled polypeptides [3]. This accounts for the lack of stereoselectivity in the oxidation reaction, which takes place at much higher rate however (Fig. 2).

Research paper thumbnail of Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis

Human Molecular Genetics, Apr 4, 2014

RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dys... more RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits and predisposition to certain malignancies, are caused by constitutional dysregulation of RAS signalling predominantly through the RAF/MEK/ERK (MAPK) cascade. We report on two germline mutations (p.Gly39dup and p.Val55Met) in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare (2 subjects among 504 individuals analysed) and variable phenotype with features partially overlapping Noonan syndrome, the most common RASopathy. We also identified somatic RRAS mutations (p.Gly39dup and p.Gln87Leu) in 2 of 110 cases of non-syndromic juvenile myelomonocytic leukaemia, a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signalling, defining an atypical form of this haematological disorder rapidly progressing to acute myeloid leukaemia. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in Caenorhabditis elegans enhanced RAS signalling and engendered protruding vulva, a phenotype previously linked to the RASopathy-causing SHOC2 S2G mutant. Overall, these findings provide evidence of a functional link between RRAS and MAPK signalling and reveal an unpredicted role of enhanced RRAS function in human disease.

Research paper thumbnail of A Nitroxide Derivative as a Probe for Conformational Studies of Short Linear Peptides in Solution. Spectroscopic and Molecular Mechanics Investigation

Journal of Physical Chemistry B, Sep 15, 1998

The photophysics of linear Aib-based hexapeptides (Aib) R-aminoisobutyric acid) of general formul... more The photophysics of linear Aib-based hexapeptides (Aib) R-aminoisobutyric acid) of general formula Ac-Toac-(Aib) n-Trp-(Aib) r-OtBu (TnTrp), where n + r) 4, and Toac and Trp are a nitroxide spin labeled C R,R-disubstituted glycine and tryptophan, respectively, were investigated in methanol and dioxane solutions by steady-state and time-resolved fluorescence measurements. Another hexapeptide, i.e., Boc-(S)Bin-Ala-Aib-Toac-(Ala) 2-OtBu (T-Bin), carrying the same nitroxide derivative and a binaphthyl as the fluorophore was also studied by the same techniques. Quenching of the excited tryptophan or binaphthyl chromophore proceeds on a time scale from subnanoseconds to a few nanoseconds, depending on the conformers distribution in solution. CD and IR spectral patterns in methanol or CDCl 3 suggest that the backbone of the peptides examined is in the 3 10-helical conformation, thus preserving the structural features of the crystal state, as earlier determined by X-ray diffraction measurements. The fluorescence results were satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited tryptophan or binaphthyl to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process. Molecular mechanics calculations coupled with time decay data allowed us to build up the most probable structures of these peptides in methanol solution.

Research paper thumbnail of Carnitine palmitoyl transferase-1A (CPT1A): a new tumor specific target in human breast cancer

Oncotarget, Jan 21, 2016

Transcriptional mechanisms epigenetically-regulated in tumoral tissues point out new targets for ... more Transcriptional mechanisms epigenetically-regulated in tumoral tissues point out new targets for anti-cancer therapies. Carnitine palmitoyl transferase I (CPT1) is the rate-limiting enzyme in the transport of long-chain fatty acids for β-oxidation. Here we identified the tumor specific nuclear CPT1A as a product of the transcript variant 2, that doesn't retain the classical transferase activity and is strongly involved in the epigenetic regulation of cancer pro-survival, cell death escaping and tumor invasion pathways. The knockdown of CPT1A variant 2 by small interfering RNAs (siRNAs), was sufficient to induce apoptosis in MCF-7, SK-BR3 and MDA-MB-231 breast cancer cells. The cell death triggered by CPT1A silencing correlated with reduction of HDAC activity and histone hyperacetylation. Docking experiments and molecular dynamics simulations confirmed an high binding affinity of the variant 2 for HDAC1. The CPT1A silenced cells showed an up-regulated transcription of pro-apoptotic genes (BAD, CASP9, COL18A1) and down-modulation of invasion and metastasis related-genes (TIMP-1, PDGF-A, SERPINB2). These findings provide evidence of the CPT1 variant 2 involvement in breast cancer survival, cell death escape and invasion. Thus, we propose nuclear CPT1A as a striking tumor specific target for anticancer therapeutics, more selective and effective as compared with the well-known HDAC inhibitors.

Research paper thumbnail of Ultrasound-Stimulated PVA Microbubbles as a Green and Handy Tool for the Cleaning of Cellulose-Based Materials

Gels, Jun 23, 2023

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Polymer-supported catalysis: stereoselective oxidation of L-dopa by iron(III) complex ions anchored to asymmetric polymers

Macromolecules, Mar 1, 1986

ABSTRACT

Research paper thumbnail of A selective chemoreceptor for Aflatoxin B1

Aflatoxin B1 (AFB1) is a toxin produced by the mould fungus varieties Aspergillus Flavus and Aspe... more Aflatoxin B1 (AFB1) is a toxin produced by the mould fungus varieties Aspergillus Flavus and Aspergillus parasiticus, which grows on foodstuffs like peanuts, flour, pepper etc. Due to its high toxicity for the human health, the AFB1 allowed concentration in food and animal feed is limited to ppb according to last EC directives [1]. At present, the methods available for aflatoxin detection are based mainly on HPLC or ELISA techniques. Therefore, new methods with low detection limit and sufficient selectivity towards AFB1 with respect to its less toxic analogous molecules, aflatoxins G1 (AFG1) and G2 (AFG2), need to be developed. Furthermore, portable and simple methods are preferred for the possibility to be used directly on site. In this respect, by using computer modelling, we have identified a chemoreceptor, based on a porphyrin moiety, able to bind selectively AFB1 with respect to AFG1. These predictions were confirmed by evaluating the binding properties of different porphyrin molecules with AFB1, AFG1 and AFG2 in THF. The system was fully characterised by means of UV absorption and steady-state and time resolved fluorescence measurements.

Research paper thumbnail of New Strategy for the Cleaning of Paper Artworks: A Smart Combination of Gels and Biosensors

Advances in chemistry, Sep 2, 2014

In this work an outlook on the design and application, in the cultural heritage field, of new too... more In this work an outlook on the design and application, in the cultural heritage field, of new tools for diagnostic and cleaning use, based on biocompatible hydrogels and electrochemical sensors, is reported. The use of hydrogels is intriguing because it does not require liquid treatment that could induce damage on artworks, while electrochemical biosensors not only are easy to prepare, but also can be selective for a specific compound and therefore are suitable for monitoring the cleaning process. In the field of restoration of paper artworks, more efforts have to be done in order to know how to perform the best way for an effective restoration. Rigid Gellan gel, made up of Gellan gum and calcium acetate, was proposed as a paper cleaning treatment, and selective biosensors for substances to be removed from this gel have been obtained by choosing the appropriate enzymes to be immobilized. Using this approach, it is possible to know when the cleanup process will be completed, avoiding lengthy and sometimes unnecessary cleaning material applications.

Research paper thumbnail of Germline Missense Mutations Affecting KRAS Isoform B Are Associated with a Severe Noonan Syndrome Phenotype

American Journal of Human Genetics, Jul 1, 2006

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphi... more Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring-binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor.

Research paper thumbnail of Conformational calculations restrained by NMR and fluorescence data on functionalized Aib-based peptides

Kluwer Academic Publishers eBooks, Dec 2, 2005

ABSTRACT

Research paper thumbnail of A New Concept: A Peptide Monolayer for Accurate Positioning of Electroactive Probes at Close Distance from a Gold Surface

Research paper thumbnail of Peptide-sandwiched protoporphyrin compounds mimicking hemoprotein structures in solution

Biopolymers, 2000

A series of covalently bound peptide-protoporphyrin-peptide compounds, also carrying naphthalene ... more A series of covalently bound peptide-protoporphyrin-peptide compounds, also carrying naphthalene (N) to allow a photophysical investigation, were synthesized. Their general formula is P(nN)(2), where P refers to protoporphyrin IX, and n to the number of amino acids in the sequence Boc-Leu-Leu-Lys-(Ala)(x) -Leu-Leu-Lys-OtBu of each backbone chain (x = 0-3; n = x + 6). Their structural features in methanol solution were investigated by ir and CD spectra, and by steady-state and time resolved fluorescence experiments as well. The ir spectra indicate that intramolecularly H-bonded conformations form, and CD data in both methanol and water-methanol mixture suggest the presence of alpha-helix structure. Quenching of excited naphthalene takes place by electronic energy transfer from singlet N* to P ground state. Fluorescence decays coupled with molecular mechanics calculations indicate that two conformers for each dimeric peptide are the major contributors to the observed phenomena. These conformers are characterized by a globular, protein-like structure, where the protoporphyrin resides in a central pocket, while the two N groups are externally situated. Of the four N linkages in the two conformers, three of them attain a very similar steric arrangement around the central P molecule, in terms of both center-to-center distance and mutual orientation, while the fourth experiences a different steric disposition as compared to the others. Experimental photophysical parameters satisfactorily compare with those obtained by theoretical calculations, within the Förster mechanism for long-range energy transfer, only when the mutual orientation of the chromophores was also taken into account. This implies that interconversion among conformational substates of probes linkages is slow on the time scale of the energy transfer process.

Research paper thumbnail of Controlling the Formation of Peptide Films: Fully Developed Helical Peptides are Required to Obtain a Homogenous Coating over a Large Area

Collection of Czechoslovak Chemical Communications, Nov 1, 2019

The influence of conformational dynamics on the self-assembly process of a novel, conformationall... more The influence of conformational dynamics on the self-assembly process of a novel, conformationally constrained, analogue of the natural, antimicrobial peptide trichogin GA IV was analysed by spectroscopic methods, microscopy imaging at nanometric resolution, and molecular dynamics simulations. The formation of antimicrobial peptide films at the air/water interface and their deposition on a graphite or a mica substrate were investigated. Combining experimental evidence with molecular dynamics simulation, we demonstrate that only the fully-developed helical structure of the analogue promotes formation of ordered aggregates nucleating the growth of micrometric rods, that give rise to homogenous coating over wide regions of the hydrophilic mica. This work proves the influence of helix flexibility on peptide self-organization and orientation on surfaces, key steps in the interaction of antimicrobial peptides with their target biological membranes.

Research paper thumbnail of Photophysical Properties of 1,3,5-Tris(2-naphthyl)benzene and Related Less-Arylated Compounds: Experimental and Theoretical Investigations

Journal of Physical Chemistry A, Aug 14, 2009

Recently, a growing interest has concerned compounds characterized by high chemical and photophys... more Recently, a growing interest has concerned compounds characterized by high chemical and photophysical stability and high quantum yield for their possible technological applications. 1,3,5-Tris(2-naphthyl)benzene (N3B), 1,3-bis(2-naphthyl)benzene (N2B), and 2-naphthyl-benzene (N1B) are promising compounds, but they needed a detailed photophysical characterization. In this context, theoretical and experimental investigations have been carried out. Steady-state and decay time fluorescence measurements indicate that the second naphthyl group, added in the meta position of N1B, perturbs the electronic levels, whereas the further naphthyl addition, leading to N3B, does not promote changes in all of the observed properties. The investigated compounds show a biexponential fluorescence decay that has been attributed to a rearrangement involving the exited states S(1) and S(2). The minimum structure corresponding to the S(1) and S(2) states has been obtained at the configuration interaction with single excitations (CIS) level of theory. For the ground-state geometry, a conformational analysis at the Hartree-Fock level has also been carried out. We have evaluated the energy gaps between electronic levels by using Zerner's intermediate neglect of differential overlap (ZINDO) method. The species involved in the fluorescence have been experimentally characterized, and the decay-associated spectra have been obtained.

Research paper thumbnail of A spectroscopic and molecular mechanics investigation on a series of AIB-based linear peptides and a peptide template, both containing tryptophan and a nitroxide derivative as probes

Biopolymers, Feb 1, 2000

Linear Aib-based hexapeptides, of the general formula Ac-Toac-(Aib)(n) -Trp-(Aib)(r) -OtBu [T(Aib... more Linear Aib-based hexapeptides, of the general formula Ac-Toac-(Aib)(n) -Trp-(Aib)(r) -OtBu [T(Aib)(n) Trp], where n + r = 4, and Toac is a nitroxide spin-labeled C(alpha,alpha)-disubstituted glycine, were investigated by steady-state and time-resolved fluorescence measurements in different solvent media. A related peptide, i.e., cyclo-¿Orn-[(Aib)(2)-Trp-(Aib)(2)-Z]-Asp-[(Aib)(2)-Toac-(Aib)(2)-+ ++OtBu ]¿ [T-cyclo-Trp], was also studied by the same techniques. It is a L-Orn, L-Asp diketopiperazine template, to which two Aib-based chains are covalently attached, each one containing one chromophore only, i.e., Trp or Toac. Whatever the solvent, in the former series of peptides quenching of the excited Trp exhibits three lifetime components and proceeds on a time scale from subnanoseconds to a few nanoseconds, while in the case of the template the same process occurs entirely on the nanoscale time scale, exhibiting two lifetimes only. The ir absorption spectral patterns suggest that the backbone of the peptides examined is in the 3(10)-helical conformation, as earlier determined by x-ray diffraction for T(Aib)(3)Trp in the crystal state. In all cases, the fluorescence results are satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited Trp to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process, allowing us to estimate the dynamics of the process. Molecular mechanics calculations coupled with time decay data made it possible to build up the most probable structures of these peptides in solution.

Research paper thumbnail of Interdisciplinary approach to develop a disposable real time monitoring tool for the cleaning of graphic artworks. Application on “le Nozze di Psiche”

Microchemical Journal, May 1, 2018

Disposable non-invasive and compatible real time monitoring tool was developed in order to follow... more Disposable non-invasive and compatible real time monitoring tool was developed in order to follow the cleaning process of paper artwork directly in situ. This tool was based on a biocompatible cleaning hydrogel coupled with flow electrochemical diagnostic tool, suitable to verify in situ and in a simple way the assessment of degradation of artwork and the efficiency of cleaning process. In this paper, the results obtained by applying this tool on a great format artwork with a lining as support, "Le Nozze di Psiche", engraved by Diana Scultori, printed in 1613, are reported. This opera was affected by a structural and chromatic deterioration due to a strong oxidative degradation. Such deterioration was probably accelerated by the adhesive (a mixture of starch paste and animal glue) used in a previous lining intervention. In this case, the cleaning agents used are rigid hydrogels of Gellan gum, modified with hydrolytic enzymes. By using the flow sampling system, all materials removed by the gel was carried up to a thin layer cell containing a selective electrochemical biosensors, suitable to monitor both treatments, the cleaning process and the removal of lining. These were monitored, allowing understanding when both processes were completed, thus avoiding lengthy and unnecessary cleaning applications. The effectiveness of cleaning with Gellan gel was assessed quantitatively by using non-invasive optical reflectance spectroscopy by a portable instrumentation, elaborating data with an improved version of the Kubelka-Munk theory in order to recover the absorption coefficient of the cellulose fibers of "Le Nozze di Psiche". The concentration of oxidized groups acting as chromophores was obtained by comparing the experimental optical absorption spectra to those simulated computationally by using TDDFT-based calculations. By following the cleaning with Gellan gel the results indicate a large decrease of the concentration of degradation product of cellulose. Moreover, chromatographic analysis were carried out in order to evaluate the amount of acid compound, produced during the aging and present on the graphic artwork, using the Gellan gel after cleaning step. The results obtained from the restoration of "Le Nozze di Psiche" have allowed the restorers to evaluate innovative methods for cleaning treatment of paper artworks with a highly specialized scientific-diagnostic approach.

Research paper thumbnail of Molecular dynamics methods to predict peptide locations in membranes: LAH4 as a stringent test case

Biochimica Et Biophysica Acta - Biomembranes, Feb 1, 2015

Determining the structure of membrane-active peptides inside lipid bilayers is essential to under... more Determining the structure of membrane-active peptides inside lipid bilayers is essential to understand their mechanism of action. Molecular dynamics simulations can easily provide atomistic details, but need experimental validation. We assessed the reliability of self-assembling (or "minimum-bias") and potential of mean force (PMF) approaches, using all-atom (AA) and coarse-grained (CG) force-fields. The LAH4 peptide was selected as a stringent test case, since it is known to attain different orientations depending on the protonation state of its four histidine residues. In all simulations the histidine side-chains inserted in the membrane when neutral, while they interacted with phospholipid headgroups in their charged state. This led to transmembrane orientations for neutral-His LAH4 in all minimum-bias AA simulations and in most CG trajectories. By contrast, the charged-His peptide stabilized membrane defects in AA simulations, whereas it was located at the membrane surface in some CG trajectories, and interacted with both lipid leaflets in others. This behavior is consistent with the higher antimicrobial activity and membrane-permeabilizing behavior of the charged-His LAH4. In addition, good agreement with solid-state NMR orientational data was observed in AA simulations. PMF calculations correctly predicted a higher membrane affinity for the neutral-His peptide. Interestingly, the structures and relative populations of PMF local free-energy minima corresponded to those determined in the less computationally demanding minimum-bias simulations. These data provide an indication about the possible membrane-perturbation mechanism of the charged-His LAH4 peptide: by interacting with lipid headgroups of both leaflets through its cationic side-chains, it could favor membrane defects and facilitate translocation across the bilayer.

Research paper thumbnail of The importance of being kinked: role of Pro residues in the selectivity of the helical antimicrobial peptide P5

Journal of Peptide Science, Oct 31, 2013

Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against... more Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against drug-resistant bacteria. Many of them kill bacteria by perturbing their membranes but exhibit no significant toxicity towards eukaryotic cells. The identification of the features responsible for this selectivity is essential for their pharmacological development. AMPs exhibit few conserved features, but a statistical analysis of an AMP sequence database indicated that many α-helical AMPs surprisingly have a helix-breaking Pro residue in the middle of their sequence. To discriminate among the different possible hypotheses for the functional role of this feature, we designed an analogue of the antimicrobial peptide P5, in which the central Pro was deleted (analogue P5Del). Pro removal resulted in a dramatic increase of toxicity. This was explained by the observation that P5Del binds both charged and neutral membranes, whereas P5 has no appreciable affinity towards neutral bilayers. CD and simulative data provided a rationalization of this behavior. In solution P5, due to the presence of Pro, attains compact conformations, in which its apolar residues are partially shielded from the solvent, whereas P5Del is more helical. These structural differences reduce the hydrophobic driving force for association of P5 to neutral membranes, whereas its binding to anionic bilayers can still take place because of electrostatic attraction. After membrane binding, the Pro residue does not preclude the attainment of a membrane-active amphiphilic helical conformation. These findings shed light on the role of Pro residues in the selectivity of AMPs and provide hints for the design of new, highly selective compounds.

Research paper thumbnail of Chiroptical, IR And Fluorescence Spectra of Short Linear Peptides in Methanol and Water-Methanol Mixtures

Springer eBooks, 1995

ABSTRACT

Research paper thumbnail of The Role of Thermodynamics in the Activity and Selectivity of Antimicrobial Peptides

Biophysical Journal, Feb 1, 2016

Research paper thumbnail of Electron transfer in catecholamines - tetrapyridine-iron(III) complex ions anchored to polypeptides

Inorganica Chimica Acta, 1983

Abstract [Fe(tetpy)(OH) 2 ] + complex ions (tetpy = 2,2′,2′',2‴-tetrapyridyl) anchored to pol... more Abstract [Fe(tetpy)(OH) 2 ] + complex ions (tetpy = 2,2′,2′',2‴-tetrapyridyl) anchored to poly(L-glutamate) (FeL) or poly(D-glutamate) (FeD) through the γ-carboxylate groups of the polymers [1] exhibit stereoselective peroxidatic activity with a number of optically active substrates, according to the reaction (pH = 7.0, Tris buffer 0.05 M ) [2, 3]: Progressive binding of complex ions was found to determine a coil-to-α-helix transition in the charged polypeptide matrices, as well as aggregation phenomena with a freezing of iron molecules inbetween helical chains. Under these conditions, electron transfer from catecholamines (L-adrenalin and L-dopa) to the central iron(III) ion, which is rate-determining, proceeds stereoselectively because extensive and possibly specific interactions between substrate molecules and the peptidic residues in the close environment of the active sites different steric constraints for the two diastereomeric precursor complexes. The rate constants of electron transfer processes can be expressed by nuclear and electronic factors which are highly sensitive to the separation of the redox centers [4]. Therefore, even small differences in steric hindrances between the diastereo-isomers would affect differently the optimal mutual orientation of the reacting OH group of the substrates and the peripheral tetrapyridyl ligand of the active sites, whose π-system very likely acts as an electron-transfer agent [5]. Preliminary results on conformational analysis of substrate-catalyst adducts support such conclusions. This is reflected in the rate constants of the electron-transfer step, as illustrated by the Lineweaver-Burk plot of the reaction catalyzed by FeL or FeD systems at a complex to polymer-residue ratio [C]/[P] = 0.20, reported in Fig. 1. From the results, the turnover numbers for the oxidation of L-adrenaline are 0.56±0.07 and 2.04±0.23 min −1 , respectively, whereas those for the oxidation of L-dopa are 0.42±0.05 and 1.14±0.12 min −1 (25.9 °C). On the other hand, the Michaelis constants are K M = (1.30±0.18) × 10 −3 and (1.05±0.15) × 10 −3 M in the former case, and (8.0±1.12) × 10 −4 and (5.50±0.48) × 10 −4 M in the latter. According to Marcus theory [6], the ultimate rate of electron transfer depends on the rate of reorganization of the surrounding medium (precursor and successor complex formation). When the asymmetric polymers play an active role in the reaction, being involved in the formation of the precursor complex the polypeptide itself might experience local conformational changes. Since the time scale of these rearrangements is unknown, it is possible that the polymer reorganization retards the overall rate of the electron transfer process. This could explain the finding that stereoselectivity occurs at the expense of the efficiency of reaction, as shown in Fig. 2. Using catalysts at low [C]/[P] ratio, the active sites are exposed to the bulk solvent and a substrate-coordinated metal chelate forms without any assistance from the coiled polypeptides [3]. This accounts for the lack of stereoselectivity in the oxidation reaction, which takes place at much higher rate however (Fig. 2).