Antonio Palleschi

Diversity and Functional Consequences of Germline and Somatic PTPN11 Mutations in Human Disease

Germline mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS) and the clinically related LEOPARD syndrome (LS), whereas somatic mutations in the same gene contribute to leukemogenesis. On the basis of our previously gathered genetic and biochemical data, we proposed a model that splits NS- and leukemia-associated PTPN11 mutations into two major classes of activating lesions with differential perturbing effects on development and hematopoiesis. To test this model, we investigated further the diversity of germline and somatic PTPN11 mutations, delineated the association of those mutations with disease, characterized biochemically a panel of mutant SHP-2 proteins recurring in NS, LS, and leukemia, and performed molecular dynamics simulations to determine the structural effects of selected mutations. Our results document a strict correlation between the identity of the lesion and disease and demonstrate that NS-causative mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated ones. Furthermore, we show that the recurrent LS-causing Y279C and T468M amino acid substitutions engender loss of SHP-2 catalytic activity, identifying a previously unrecognized behavior for this class of missense PTPN11 mutations.

Germline missense mutations affecting KRAS isoform B are associated with a severe noonan syndrome phenotype

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.

Scleroglucan: A Versatile Polysaccharide for Modified Drug Delivery

Scleroglucan is a natural polysaccharide, produced by fungi of the genus Sclerotium, that has been extensively studied for various commercial applications (secondary oil recovery, ceramic glazes, food, paints, etc.) and also shows several interesting pharmacological properties. This review focuses its attention on the use of scleroglucan, and some derivatives, in the field of pharmaceutics and in particular for the formulation of modified-release dosage forms. The reported investigations refer mainly to the following topics: natural scleroglucan suitable for the preparation of sustained release tablets and ocular formulations; oxidized and crosslinked scleroglucan used as a matrix for dosage forms sensitive to environmental conditions; co-crosslinked scleroglucan/gellan whose delivery rate can be affected by calcium ions. Furthermore, a novel hydrogel obtained with this polysaccharide and borate ions is described, and the particular structure of this hydrogel network has been interpreted in terms of conformational analysis and molecular dynamics. Profound attention is devoted to the mechanisms involved in drug release from the tested dosage forms that depend, according to the specific preparation, on swelling and/or diffusion. Experimental data are also discussed on the basis of a mathematical approach that allows a better understanding of the behavior of the tested polymeric materials.

A theoretical model of DNA curvature.

Distortions from the uniform idealized B-DNA structure are investigated in terms of differential interactions between adjacent nucleotide pairs on the basis of conformational energy calculations. A theoretical model of DNA curvature is proposed based on the evaluation of the curvature vector defined in the complex plane and the corresponding variance. The model appears to contain the basic physical features for translating the deterministic fluctuations of DNA sequences in superstructure elements. It allows the quantitative reproduction of all the available gel electrophoresis experiments on both periodical polynucleotides and tracts of DNAs as well as the theoretical prediction of the sequence dependent DNA writhing in good agreement with the experimental data. The general pattern of agreement between the theoretical and experimental data and the biological significance of the results obtained allow an extensive application of the model for the screening of DNA regions which are possible candidates for protein recognition.

Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis

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 SHOC2S2G 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.

Aggregation and water-membrane partition as major determinants of the activity of the antibiotic peptide trichogin GA IV.

Water-membrane partition and aggregation behavior are fundamental aspects of the biological activity of antibiotic peptides, natural compounds causing the death of pathogenic organisms by perturbing the permeability of their membranes. A synthetic fluorescent analog of the natural lipopeptaibol trichogin GA IV was used to study its interaction with model membranes. Time-resolved fluorescence data show that in water, an equilibrium between monomers and small aggregates is present, the two species having different affinity for membranes. Therefore, association curves are strongly dependent on peptide concentration. A similar heterogeneity is present in the membrane phase, which strongly suggests the occurrence of a monomer-aggregate equilibrium in this case, too. The relative population of each species was determined and a strong correlation between the concentration of membrane-bound aggregates and membrane leakage was found, thereby suggesting that liposome perturbation is due to peptide aggregates only. Light-scattering measurements demonstrate that leakage is not due to liposome micellization. Moreover, experiments with markers of different sizes show that molecules with a diameter of approximately 4 nm are released only to a minor extent. Overall, these results suggest that, within the concentration range explored, pore formation by peptide aggregates is the most likely mechanism of action for trichogin in membranes.

Different mechanisms of action of antimicrobial peptides: insights from fluorescence spectroscopy experiments and molecular dynamics simulations†

Most antimicrobial peptides exert their activity by interacting with bacterial membranes, thus perturbing their permeability. They are investigated as a possible solution to the insurgence of bacteria resistant to the presently available antibiotic drugs. However, several different models have been proposed for their mechanism of membrane perturbation, and the molecular details of this process are still debated. Here, we compare fluorescence spectroscopy experiments and molecular dynamics (MD) simulations regarding the association with lipid bilayers and lipid perturbation for two different amphiphilic helical antimicrobial peptides, PMAP‐23 and trichogin GA IV. PMAP‐23, a cationic peptide member of the cathelicidin family, is considered to induce membrane permeability according to the Shai‐Matsuzaki‐Huang “carpet” model, while trichogin GA IV is a neutral peptide, member of the peptaibol family. Although several lines of evidence suggest a “barrel‐stave” mechanism of pore formation for the latter peptide, its length is only half the normal thickness of a lipid bilayer. Both fluorescence spectroscopy experiments and MD simulations indicated that PMAP‐23 associates with membranes close to their surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. By contrast, trichogin GA IV can undergo a transition from a surface‐bound state to a transmembrane orientation. In the first arrangement, it does not cause any strong membrane perturbation, while in the second orientation it might be able to span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. Copyright

Diverse driving forces underlie the invariant occurrence of the T42A, E139D, I282V and T468M SHP2 amino acid substitutions causing Noonan and LEOPARD syndromes

Missense PTPN11 mutations cause Noonan and LEOPARD syndromes (NS and LS), two developmental disorders with pleiomorphic phenotypes. PTPN11 encodes SHP2, an SH2 domain-containing protein tyrosine phosphatase functioning as a signal transducer. Generally, different substitutions of a particular amino acid residue are observed in these diseases, indicating that the crucial factor is the residue being replaced. For a few codons, only one substitution is observed, suggesting the possibility of specific roles for the residue introduced. We analyzed the biochemical behavior and ligand-binding properties of all possible substitutions arising from single-base changes affecting codons 42, 139, 279, 282 and 468 to investigate the mechanisms underlying the invariant occurrence of the T42A, E139D and I282V substitutions in NS and the Y279C and T468M changes in LS. Our data demonstrate that the isoleucine-to-valine change at codon 282 is the only substitution at that position perturbing the stability of SHP2s closed conformation without impairing catalysis, while the threonine-to-alanine change at codon 42, but not other substitutions of that residue, promotes increased phosphopeptide-binding affinity. The recognition specificity of the C-SH2 domain bearing the E139D substitution differed substantially from its wild-type counterpart acquiring binding properties similar to those observed for the N-SH2 domain, revealing a novel mechanism of SHP2s functional dysregulation. Finally, while functional selection does not seem to occur for the substitutions at codons 279 and 468, we point to deamination of the methylated cytosine at nucleotide 1403 as the driving factor leading to the high prevalence of the T468M change in LS.

Structural and rheological characterization of Scleroglucan/borax hydrogel for drug delivery

The polysaccharide Scleroglucan, one of the most rigid polymers found in nature, can form a chemical/physical gel, in the presence of borax. The obtained hydrogel was loaded with three different model molecules (Theophylline, Vitamin B12 and Myoglobin) and then, after freeze-drying, was used as a matrix for tablets. The release profiles of the substances from the dosage forms were evaluated; the matrix appeared capable to modulate the diffusion of the chosen molecules, and different diffusion rates were observed, according to the different radii of the tested molecules. Interestingly, in the dissolution medium the matrix undergoes an anisotropic swelling taking place only in the axial direction, while a negligible radial variation occurs. The water uptake of the matrix occurs according to a Fickian process. Samples at two different polymer concentrations (0.7 and 2.3%, w/v) were characterized in terms of rheological and mechanical parameters and the properties were interpreted in terms of the molecular structure obtained by conformational analysis. The flow curves acquired in the viscoelasticity interval, show the effect of the borate ion in improving the resistance of the gel in comparison to the polymer alone. The evaluation of the moduli indicates that the system is viscoelastic, with an appreciable liquid component that increases as the polymer concentration decreases. Also the cohesion of the gel is higher in comparison to the Scleroglucan and is strongly dependent on temperature. The combination of experimental and theoretical conformational analysis approaches, allowed us to propose a model for the structure of the macromolecular network and to give an explanation to the anomalous swelling that was observed. It came out that the polymer can built up a channel structure, mediated via borax ion interaction, that can accommodate guest molecules of different size.

Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.