Publications by authors named "Gianfranco Bocchinfuso"

51 Publications

Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation.

J Pept Sci 2021 Jun 10:e3356. Epub 2021 Jun 10.

PEPSA-LAB, Department of Chemical Sciences and Technology, University of Rome Tor Vergata, Rome, Italy.

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH ), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.
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http://dx.doi.org/10.1002/psc.3356DOI Listing
June 2021

Compound heterozygosity for PTPN11 variants in a subject with Noonan syndrome provides insights into the mechanism of SHP2-related disorders.

Clin Genet 2021 03 4;99(3):457-461. Epub 2021 Jan 4.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

The RASopathies are a family of clinically related disorders caused by mutations affecting genes participating in the RAS-MAPK signaling cascade. Among them, Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML) are allelic conditions principally associated with dominant mutations in PTPN11, which encodes the nonreceptor SH2 domain-containing protein tyrosine phosphatase SHP2. Individual PTPN11 mutations are specific to each syndrome and have opposite consequences on catalysis, but all favor SHP2's interaction with signaling partners. Here, we report on a subject with NS harboring biallelic variants in PTPN11. While the former (p.Leu261Phe) had previously been reported in NS, the latter (p.Thr357Met) is a novel change impairing catalysis. Members of the family carrying p.Thr357Met, however, did not show any obvious feature fitting NSML or within the RASopathy phenotypic spectrum. A major impact of this change on transcript processing and protein stability was excluded. These findings further support the view that NSML cannot be ascribed merely to impaired SHP2's catalytic activity and suggest that PTPN11 mutations causing this condition act through an alternative dominant mechanism.
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http://dx.doi.org/10.1111/cge.13904DOI Listing
March 2021

A Recurrent Gain-of-Function Mutation in CLCN6, Encoding the ClC-6 Cl/H-Exchanger, Causes Early-Onset Neurodegeneration.

Am J Hum Genet 2020 12 19;107(6):1062-1077. Epub 2020 Nov 19.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), 13125 Berlin, Germany; Max-Delbrück-Centrum für Molekulare Medizin (MDC), 13125 Berlin, Germany; NeuroCure Cluster of Excellence, Charité Universitätsmedizin, 10117 Berlin, Germany. Electronic address:

Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl/H exchangers function as electric shunts for proton pumping and in luminal Cl accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl/H-exchanger ClC-6. Whereas Clcn6 mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6, but not ClC-6, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl/H exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6 or ClC-6 generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H-driven Cl accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.
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http://dx.doi.org/10.1016/j.ajhg.2020.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820737PMC
December 2020

Aggregation propensity of therapeutic fibrin-homing pentapeptides: insights from experiments and molecular dynamics simulations.

Soft Matter 2020 Nov;16(44):10169-10179

Department of Chemical Engineering and Barcelona Research Center in Multiscale Science and Engineering, Universitat Polytècnica de Catalunya, 08019 Barcelona, Spain.

CREKA (Cys-Arg-Glu-Lys-Ala) and its engineered analogue CRMeEKA, in which Glu has been replaced by N-methyl-Glu to provide resistance against proteolysis, are emerging pentapeptides that were specifically designed to bind fibrin-fibronectin complexes accumulated in the walls of tumour vessels. However, many of the intrinsic properties of CREKA and CRMeEKA, which are probably responsible for their different behaviour when combined with other materials (such as polymers) for diagnosis and therapeutics, remain unknown yet. The intrinsic tendency of these pentapeptides to form aggregates has been analysed by combining experimental techniques and atomistic Molecular Dynamics (MD) simulations. Dynamic light scattering assays show the formation of nanoaggregates that increase in size with the peptide concentration, even though aggregation occurs sooner for CRMeEKA, independently of the peptide concentration. FTIR and circular dichroism spectroscopy studies suggest that aggregated pentapeptides do not adopt any secondary structure. Atomistic MD trajectories show that CREKA aggregates faster and forms bigger molecular clusters than CRMeEKA. This behaviour has been explained by stability of the conformations adopted by un-associated peptide strands. While CREKA molecules organize by forming intramolecular backbone - side chain hydrogen bonds, CRMeEKA peptides display main chain - main chain hydrogen bonds closing very stable γ- or β-turns. Besides, energetic analyses reveal that CRMeEKA strands are better solvated in water than CREKA ones, independent of whether they are assembled or un-associated.
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http://dx.doi.org/10.1039/d0sm00930jDOI Listing
November 2020

Enhanced MAPK1 Function Causes a Neurodevelopmental Disorder within the RASopathy Clinical Spectrum.

Am J Hum Genet 2020 09 27;107(3):499-513. Epub 2020 Jul 27.

Institute of Human Genetics, University Hospital Magdeburg, 39120 Magdeburg, Germany.

Signal transduction through the RAF-MEK-ERK pathway, the first described mitogen-associated protein kinase (MAPK) cascade, mediates multiple cellular processes and participates in early and late developmental programs. Aberrant signaling through this cascade contributes to oncogenesis and underlies the RASopathies, a family of cancer-prone disorders. Here, we report that de novo missense variants in MAPK1, encoding the mitogen-activated protein kinase 1 (i.e., extracellular signal-regulated protein kinase 2, ERK2), cause a neurodevelopmental disease within the RASopathy phenotypic spectrum, reminiscent of Noonan syndrome in some subjects. Pathogenic variants promote increased phosphorylation of the kinase, which enhances translocation to the nucleus and boosts MAPK signaling in vitro and in vivo. Two variant classes are identified, one of which directly disrupts binding to MKP3, a dual-specificity protein phosphatase negatively regulating ERK function. Importantly, signal dysregulation driven by pathogenic MAPK1 variants is stimulus reliant and retains dependence on MEK activity. Our data support a model in which the identified pathogenic variants operate with counteracting effects on MAPK1 function by differentially impacting the ability of the kinase to interact with regulators and substrates, which likely explains the minor role of these variants as driver events contributing to oncogenesis. After nearly 20 years from the discovery of the first gene implicated in Noonan syndrome, PTPN11, the last tier of the MAPK cascade joins the group of genes mutated in RASopathies.
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http://dx.doi.org/10.1016/j.ajhg.2020.06.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477014PMC
September 2020

Structural Determinants of Phosphopeptide Binding to the N-Terminal Src Homology 2 Domain of the SHP2 Phosphatase.

J Chem Inf Model 2020 06 29;60(6):3157-3171. Epub 2020 May 29.

Department of Chemical Science and Technologies, University of Rome Tor Vergata, 00133, Rome, Italy.

SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by , plays a fundamental role in the modulation of several signaling pathways. Germline and somatic mutations in are associated with different rare diseases and hematologic malignancies, and recent studies have individuated SHP2 as a central node in oncogenesis and cancer drug resistance. The SHP2 structure includes two Src homology 2 domains (N-SH2 and C-SH2) followed by a catalytic protein tyrosine phosphatase (PTP) domain. Under basal conditions, the N-SH2 domain blocks the active site, inhibiting phosphatase activity. Association of the N-SH2 domain with binding partners containing short amino acid motifs comprising a phosphotyrosine residue (pY) leads to N-SH2/PTP dissociation and SHP2 activation. Considering the relevance of SHP2 in signaling and disease and the central role of the N-SH2 domain in its allosteric regulation mechanism, we performed microsecond-long molecular dynamics (MD) simulations of the N-SH2 domain complexed to 12 different peptides to define the structural and dynamical features determining the binding affinity and specificity of the domain. Phosphopeptide residues at position -2 to +5, with respect to pY, have significant interactions with the SH2 domain. In addition to the strong interaction of the pY residue with its conserved binding pocket, the complex is stabilized hydrophobically by insertion of residues +1, +3, and +5 in an apolar groove of the domain and interaction of residue -2 with both the pY and a protein surface residue. Additional interactions are provided by hydrogen bonds formed by the backbone of residues -1, +1, +2, and +4. Finally, negatively charged residues at positions +2 and +4 are involved in electrostatic interactions with two lysines (Lys89 and Lys91) specific for the SHP2 N-SH2 domain. Interestingly, the MD simulations illustrated a previously undescribed conformational flexibility of the domain, involving the core β sheet and the loop that closes the pY binding pocket.
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http://dx.doi.org/10.1021/acs.jcim.0c00307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007070PMC
June 2020

Co-occurring WARS2 and CHRNA6 mutations in a child with a severe form of infantile parkinsonism.

Parkinsonism Relat Disord 2020 03 15;72:75-79. Epub 2020 Feb 15.

Department of Physiology and Pharmacology, Sapienza University of Rome, Rome, Italy; IRCCS Neuromed, Pozzilli, IS, Italy.

Objective: To investigate the molecular cause(s) underlying a severe form of infantile-onset parkinsonism and characterize functionally the identified variants.

Methods: A trio-based whole exome sequencing (WES) approach was used to identify the candidate variants underlying the disorder. In silico modeling, and in vitro and in vivo studies were performed to explore the impact of these variants on protein function and relevant cellular processes.

Results: WES analysis identified biallelic variants in WARS2, encoding the mitochondrial tryptophanyl tRNA synthetase (mtTrpRS), a gene whose mutations have recently been associated with multiple neurological phenotypes, including childhood-onset, levodopa-responsive or unresponsive parkinsonism in a few patients. A substantial reduction of mtTrpRS levels in mitochondria and reduced OXPHOS function was demonstrated, supporting their pathogenicity. Based on the infantile-onset and severity of the phenotype, additional variants were considered as possible genetic modifiers. Functional assessment of a selected panel of candidates pointed to a de novo missense mutation in CHRNA6, encoding the α6 subunit of neuronal nicotinic receptors, which are involved in the cholinergic modulation of dopamine release in the striatum, as a second event likely contributing to the phenotype. In silico, in vitro (Xenopus oocytes and GH4C1 cells) and in vivo (C. elegans) analyses demonstrated the disruptive effects of the mutation on acetylcholine receptor structure and function.

Conclusion: Our findings consolidate the association between biallelic WARS2 mutations and movement disorders, and suggest CHRNA6 as a genetic modifier of the phenotype.
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http://dx.doi.org/10.1016/j.parkreldis.2020.02.003DOI Listing
March 2020

Sound-driven dissipative self-assembly of aromatic biomolecules into functional nanoparticles.

Nanoscale Horiz 2020 03;5(3):553-563

Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma "Tor Vergata", via della ricerca scientifica 1, 00133, Rome, Italy and Department of Chemical Engineering, University of Melbourne, VIC 3010, Australia.

Dissipative self-assembly processes were recently exploited to assemble synthetic materials into supramolecular structures. In most cases, chemical fuel or light driven self-assembly of synthetic molecules was reported. Herein, experimental and computational approaches were used to unveil the role of acoustic cavitation in the formation of supramolecular nanoaggregates by dissipative self-assembly. Acoustic cavitation bubbles were employed as an energy source and a transient interface to fuel and refuel the dissipative self-assembly of simple aromatic biomolecules into uniform nanoparticles. Molecular dynamics simulations were applied to predict the formation of metastable aggregates and the dynamic exchange of the interacting molecules in the nanoaggregates. The intracellular trafficking and dissipative dissolution of the nanoparticles were tracked by microscopy imaging.
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http://dx.doi.org/10.1039/c9nh00611gDOI Listing
March 2020

Pathogenic PTPN11 variants involving the poly-glutamine Gln -Gln -Gln stretch highlight the relevance of helix B in SHP2's functional regulation.

Hum Mutat 2020 06 11;41(6):1171-1182. Epub 2020 Mar 11.

Institute of Human Genetics, University Hospital of Magdeburg, Otto-von-Guericke-University, Magdeburg, Germany.

Germline PTPN11 mutations cause Noonan syndrome (NS), the most common disorder among RASopathies. PTPN11 encodes SHP2, a protein tyrosine-phosphatase controlling signaling through the RAS-MAPK and PI3K-AKT pathways. Generally, NS-causing PTPN11 mutations are missense changes destabilizing the inactive conformation of the protein or enhancing its binding to signaling partners. Here, we report on two PTPN11 variants resulting in the deletion or duplication of one of three adjacent glutamine residues (Gln -to-Gln ). While p.(Gln257dup) caused a typical NS phenotype in carriers of a first family, p.(Gln257del) had incomplete penetrance in a second family. Missense mutations involving Gln had previously been reported in NS. This poly-glutamine stretch is located on helix B of the PTP domain, a region involved in stabilizing SHP2 in its autoinhibited state. Molecular dynamics simulations predicted that changes affecting this motif perturb the SHP2's catalytically inactive conformation and/or substrate recognition. Biochemical data showed that duplication and deletion of Gln variably enhance SHP2's catalytic activity, while missense changes involving Gln affect substrate specificity. Expression of mutants in HEK293T cells documented their activating role on MAPK signaling, uncoupling catalytic activity and modulation of intracellular signaling. These findings further document the relevance of helix B in the regulation of SHP2's function.
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http://dx.doi.org/10.1002/humu.24007DOI Listing
June 2020

Rational Design of Antiangiogenic Helical Oligopeptides Targeting the Vascular Endothelial Growth Factor Receptors.

Front Chem 2019 29;7:170. Epub 2019 Mar 29.

Department of Chemical Science and Technologies, University of Rome Tor Vergata, Rome, Italy.

Tumor angiogenesis, essential for cancer development, is regulated mainly by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which are overexpressed in cancer cells. Therefore, the VEGF/VEGFR interaction represents a promising pharmaceutical target to fight cancer progression. The VEGF surface interacting with VEGFRs comprises a short α-helix. In this work, helical oligopeptides mimicking the VEGF-C helix were rationally designed based on structural analyses and computational studies. The helical conformation was stabilized by optimizing intramolecular interactions and by introducing helix-inducing C-disubstituted amino acids. The conformational features of the synthetic peptides were characterized by circular dichroism and nuclear magnetic resonance, and their receptor binding properties and antiangiogenic activity were determined. The best hits exhibited antiangiogenic activity at nanomolar concentrations and were resistant to proteolytic degradation.
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http://dx.doi.org/10.3389/fchem.2019.00170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449863PMC
March 2019

Mutations in KCNK4 that Affect Gating Cause a Recognizable Neurodevelopmental Syndrome.

Am J Hum Genet 2018 10;103(4):621-630

Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy. Electronic address:

Aberrant activation or inhibition of potassium (K) currents across the plasma membrane of cells has been causally linked to altered neurotransmission, cardiac arrhythmias, endocrine dysfunction, and (more rarely) perturbed developmental processes. The K channel subfamily K member 4 (KCNK4), also known as TRAAK (TWIK-related arachidonic acid-stimulated K channel), belongs to the mechano-gated ion channels of the TRAAK/TREK subfamily of two-pore-domain (K2P) K channels. While K2P channels are well known to contribute to the resting membrane potential and cellular excitability, their involvement in pathophysiological processes remains largely uncharacterized. We report that de novo missense mutations in KCNK4 cause a recognizable syndrome with a distinctive facial gestalt, for which we propose the acronym FHEIG (facial dysmorphism, hypertrichosis, epilepsy, intellectual disability/developmental delay, and gingival overgrowth). Patch-clamp analyses documented a significant gain of function of the identified KCNK4 channel mutants basally and impaired sensitivity to mechanical stimulation and arachidonic acid. Co-expression experiments indicated a dominant behavior of the disease-causing mutations. Molecular dynamics simulations consistently indicated that mutations favor sealing of the lateral intramembrane fenestration that has been proposed to negatively control K flow by allowing lipid access to the central cavity of the channel. Overall, our findings illustrate the pleiotropic effect of dysregulated KCNK4 function and provide support to the hypothesis of a gating mechanism based on the lateral fenestrations of K2P channels.
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http://dx.doi.org/10.1016/j.ajhg.2018.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174320PMC
October 2018

Clinical and functional characterization of two novel ZBTB20 mutations causing Primrose syndrome.

Hum Mutat 2018 07 17;39(7):959-964. Epub 2018 May 17.

Institute of Medical Genetics, University of Zurich, Schlieren-Zurich, Switzerland.

Primrose syndrome (PS) is a rare disorder characterized by macrocephaly, tall stature, intellectual disability, autistic traits, and disturbances of glucose metabolism with insulin-resistant diabetes and distal muscle wasting occurring in adulthood. The disorder is caused by functional dysregulation of ZBTB20, a transcriptional repressor controlling energetic metabolism and developmental programs. ZBTB20 maps in a genomic region that is deleted in the 3q13.31 microdeletion syndrome, which explains the clinical overlap between the two disorders. A narrow spectrum of amino acid substitutions in a restricted region of ZBTB20 encompassing the first and second zinc-finger motifs have been reported thus far. Here, we characterize clinically and functionally the first truncating mutation [(c.1024delC; p.(Gln342Serfs*42)] and a missense change affecting the third zinc-finger motif of the protein [(c.1931C > T; p.(Thr644Ile)]. Our data document that both mutations have dominant negative impact on wild-type ZBTB20, providing further evidence of the specific behavior of PS-causing mutations on ZBTB20 function.
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http://dx.doi.org/10.1002/humu.23546DOI Listing
July 2018

Selectively targeting bacteria by tuning the molecular design of membrane-active peptidomimetic amphiphiles.

Chem Commun (Camb) 2018 May;54(39):4943-4946

Antimicrobial Research Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, Karnataka, India.

Here we report the design of membrane-active peptidomimetic molecules with a tunable arrangement of hydrophobic and polar groups. In spite of having the same chemical composition, the effective hydrophobicities of the compounds were different as a consequence of their chemical structure and conformational properties. The compound with lower effective hydrophobicity demonstrated antibacterial activity that was highly selective towards bacteria over mammalian cells. This study, highlighting the role in membrane selectivity of the specific arrangement of the different moieties in the molecular structure, provides useful indications for developing non-toxic antibacterial agents.
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http://dx.doi.org/10.1039/c8cc01926fDOI Listing
May 2018

Orienting proteins by nanostructured surfaces: evidence of a curvature-driven geometrical resonance.

Nanoscale 2018 Apr;10(16):7544-7555

Laboratory for Molecular Surfaces and Nanotechnology (LAMSUN), Department of Chemical Sciences, University of Catania, Viale A.Doria 6, 95125 Catania, Italy.

Experimental and theoretical reports have shown that nanostructured surfaces have a dramatic effect on the amount of protein adsorbed and the conformational state and, in turn, on the performances of the related devices in tissue engineering strategies. Here we report an innovative method to prepare silica-based nanostructured surfaces with a reproducible, well-defined local curvature, consisting of ordered hexagonally packed arrays of curved hemispheres, from nanoparticles of different diameters (respectively 147 nm, 235 nm and 403 nm). The nanostructured surfaces have been made chemically homogeneous by partially embedding silica nanoparticles in poly(hydroxymethylsiloxane) films, further modified by means of UV-O3 treatments. This paper has been focused on the experimental and theoretical study of laminin, taken as a model protein, to study the nanocurvature effects on the protein configuration at nanostructured surfaces. A simple model, based on the interplay of electrostatic interactions between the charged terminal domains of laminin and the nanocurved charged surfaces, closely reproduces the experimental findings. In particular, the model suggests that nanocurvature drives the orientation of rigid proteins by means of a "geometrical resonance" effect, involving the matching of dimensions, charge distribution and spatial arrangement of both adsorbed molecules and adsorbent nanostructures. Overall, the results pave the way to unravel the nanostructured surface effects on the intra- and inter-molecular organization processes of proteins.
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http://dx.doi.org/10.1039/c8nr00037aDOI Listing
April 2018

Enhanced EGFR Targeting Activity of Plasmonic Nanostructures with Engineered GE11 Peptide.

Adv Healthc Mater 2017 Dec 25;6(23). Epub 2017 Sep 25.

Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131, Padova, Italy.

Plasmonic nanostructures show important properties for biotechnological applications, but they have to be guided on the target for exploiting their potentialities. Antibodies are the natural molecules for targeting. However, their possible adverse immunogenic activity and their cost have suggested finding other valid substitutes. Small molecules like peptides can be an alternative source of targeting agents, even if, as single molecules, their binding affinity is usually not very good. GE11 is a small dodecapeptide with specific binding to the epidermal growth factor receptor (EGFR) and low immunogenicity. The present work shows that thousands of polyethylene glycol (PEG) chains modified with lysines and functionalized with GE11 on clusters of naked gold nanoparticles, obtained by laser ablation in water, achieves a better targeting activity than that recorded with nanoparticles decorated with the specific anti-EGFR antibody Cetuximab (C225). The insertion of the cationic spacer between the polymeric part of the ligand and the targeting peptide allows for a proper presentation of GE11 on the surface of the nanosystems. Surface enhanced resonance Raman scattering signals of the plasmonic gold nanoparticles are used for quantifying the targeting activity. Molecular dynamic calculations suggest that subtle differences in the exposition of the peptide on the PEG sea are important for the targeting activity.
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http://dx.doi.org/10.1002/adhm.201700596DOI Listing
December 2017

Molecular Dynamics Simulations of the Host Defense Peptide Temporin L and Its Q3K Derivative: An Atomic Level View from Aggregation in Water to Bilayer Perturbation.

Molecules 2017 Jul 22;22(7). Epub 2017 Jul 22.

Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma "Tor Vergata", Rome 00133, Italy.

Temporin L (TempL) is a 13 residue Host Defense Peptide (HDP) isolated from the skin of frogs. It has a strong affinity for lipopolysaccharides (LPS), which is related to its high activity against Gram-negative bacteria and also to its strong tendency to neutralize the pro-inflammatory response caused by LPS release from inactivated bacteria. A designed analog with the Q3K substitution shows an enhancement in both these activities. In the present paper, Molecular Dynamics (MD) simulations have been used to investigate the origin of these improved properties. To this end, we have studied the behavior of the peptides both in water solution and in the presence of LPS lipid-A bilayers, demonstrating that the main effect through which the Q3K substitution improves the peptide activities is the destabilization of peptide aggregates in water.
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http://dx.doi.org/10.3390/molecules22071235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152314PMC
July 2017

The Influence of pH on the Scleroglucan and Scleroglucan/Borax Systems.

Molecules 2017 Mar 9;22(3). Epub 2017 Mar 9.

Department of Drug Chemistry and Technologies, "Sapienza", University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.

The effects that an increase of environmental pH has on the triple helix of scleroglucan (Sclg) and on the Sclg/borax hydrogel are reported. Rheological experiments show that the hydrogel is less sensitive to pH increase than Sclg alone, while at pH = 14 a dramatic viscosity decrease takes place for both systems. This effect is evidenced also by the reduced water uptake and anisotropic elongation detected, at pH = 14, by the swelling behaviour of tablets prepared with the Sclg/borax system. On the opposite, a different behaviour was observed with guar gum and locust bean gum tablets, tested as reference polysaccharides. The effect of pH on the structure of Sclg and Sclg/borax was investigated also by means of spectroscopic approaches based on the interaction between Congo red (CR) and the Sclg triple helix. Obtained results indicated that the CR absorbance maximum is shifted as a function of pH and by the presence of borax. Principal component analysis allowed very precise identification of the pH value at which the Sclg helix collapses. Molecular dynamics simulations of the Sclg/borax-CR complex indicated that, at physiological pH, only a few ordered configurations are populated, according to the induced circular dichroism (CD) spectrum evidence.
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http://dx.doi.org/10.3390/molecules22030435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155179PMC
March 2017

Structural, Functional, and Clinical Characterization of a Novel PTPN11 Mutation Cluster Underlying Noonan Syndrome.

Hum Mutat 2017 04 7;38(4):451-459. Epub 2017 Feb 7.

Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy.

Germline mutations in PTPN11, the gene encoding the Src-homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP2), cause Noonan syndrome (NS), a relatively common, clinically variable, multisystem disorder. Here, we report on the identification of five different PTPN11 missense changes affecting residues Leu , Leu , and Arg in 16 unrelated individuals with clinical diagnosis of NS or with features suggestive for this disorder, specifying a novel disease-causing mutation cluster. Expression of the mutant proteins in HEK293T cells documented their activating role on MAPK signaling. Structural data predicted a gain-of-function role of substitutions at residues Leu and Arg exerted by disruption of the N-SH2/PTP autoinhibitory interaction. Molecular dynamics simulations suggested a more complex behavior for changes affecting Leu , with possible impact on SHP2's catalytic activity/selectivity and proper interaction of the PTP domain with the regulatory SH2 domains. Consistent with that, biochemical data indicated that substitutions at codons 262 and 265 increased the catalytic activity of the phosphatase, while those affecting codon 261 were only moderately activating but impacted substrate specificity. Remarkably, these mutations underlie a relatively mild form of NS characterized by low prevalence of cardiac defects, short stature, and cognitive and behavioral issues, as well as less evident typical facial features.
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http://dx.doi.org/10.1002/humu.23175DOI Listing
April 2017

Carnitine palmitoyl transferase-1A (CPT1A): a new tumor specific target in human breast cancer.

Oncotarget 2016 Apr;7(15):19982-96

Department of Biomedicine and Prevention, Tor Vergata University of Rome, Rome, Italy.

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.
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http://dx.doi.org/10.18632/oncotarget.6964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991433PMC
April 2016

Activating Mutations Affecting the Dbl Homology Domain of SOS2 Cause Noonan Syndrome.

Hum Mutat 2015 Nov 3;36(11):1080-7. Epub 2015 Aug 3.

Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, 00165, Italy.

The RASopathies constitute a family of autosomal-dominant disorders whose major features include facial dysmorphism, cardiac defects, reduced postnatal growth, variable cognitive deficits, ectodermal and skeletal anomalies, and susceptibility to certain malignancies. Noonan syndrome (NS), the commonest RASopathy, is genetically heterogeneous and caused by functional dysregulation of signal transducers and regulatory proteins with roles in the RAS/extracellular signal-regulated kinase (ERK) signal transduction pathway. Mutations in known disease genes account for approximately 80% of affected individuals. Here, we report that missense mutations altering Son of Sevenless, Drosophila, homolog 2 (SOS2), which encodes a RAS guanine nucleotide exchange factor, occur in a small percentage of subjects with NS. Four missense mutations were identified in five unrelated sporadic cases and families transmitting NS. Disease-causing mutations affected three conserved residues located in the Dbl homology (DH) domain, of which two are directly involved in the intramolecular binding network maintaining SOS2 in its autoinhibited conformation. All mutations were found to promote enhanced signaling from RAS to ERK. Similar to NS-causing SOS1 mutations, the phenotype associated with SOS2 defects is characterized by normal development and growth, as well as marked ectodermal involvement. Unlike SOS1 mutations, however, those in SOS2 are restricted to the DH domain.
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http://dx.doi.org/10.1002/humu.22834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604019PMC
November 2015

Mutations in KCNH1 and ATP6V1B2 cause Zimmermann-Laband syndrome.

Nat Genet 2015 Jun 27;47(6):661-7. Epub 2015 Apr 27.

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Zimmermann-Laband syndrome (ZLS) is a developmental disorder characterized by facial dysmorphism with gingival enlargement, intellectual disability, hypoplasia or aplasia of nails and terminal phalanges, and hypertrichosis. We report that heterozygous missense mutations in KCNH1 account for a considerable proportion of ZLS. KCNH1 encodes the voltage-gated K(+) channel Eag1 (Kv10.1). Patch-clamp recordings showed strong negative shifts in voltage-dependent activation for all but one KCNH1 channel mutant (Gly469Arg). Coexpression of Gly469Arg with wild-type KCNH1 resulted in heterotetrameric channels with reduced conductance at positive potentials but pronounced conductance at negative potentials. These data support a gain-of-function effect for all ZLS-associated KCNH1 mutants. We also identified a recurrent de novo missense change in ATP6V1B2, encoding the B2 subunit of the multimeric vacuolar H(+) ATPase, in two individuals with ZLS. Structural analysis predicts a perturbing effect of the mutation on complex assembly. Our findings demonstrate that KCNH1 mutations cause ZLS and document genetic heterogeneity for this disorder.
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http://dx.doi.org/10.1038/ng.3282DOI Listing
June 2015

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

Am J Hum Genet 2015 May 9;96(5):816-25. Epub 2015 Apr 9.

Center for Pediatric Research, A.I. duPont Hospital for Children, Wilmington, DE 19803, USA.

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.
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http://dx.doi.org/10.1016/j.ajhg.2015.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4570552PMC
May 2015

Mutations in ZBTB20 cause Primrose syndrome.

Nat Genet 2014 Aug 13;46(8):815-7. Epub 2014 Jul 13.

1] Department of Clinical Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. [2] Department of Pediatrics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. [3].

Primrose syndrome and 3q13.31 microdeletion syndrome are clinically related disorders characterized by tall stature, macrocephaly, intellectual disability, disturbed behavior and unusual facial features, with diabetes, deafness, progressive muscle wasting and ectopic calcifications specifically occurring in the former. We report that missense mutations in ZBTB20, residing within the 3q13.31 microdeletion syndrome critical region, underlie Primrose syndrome. This finding establishes a genetic link between these disorders and delineates the impact of ZBTB20 dysregulation on development, growth and metabolism.
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http://dx.doi.org/10.1038/ng.3035DOI Listing
August 2014

Aggregation propensity of Aib homo-peptides of different length: an insight from molecular dynamics simulations.

J Pept Sci 2014 Jul 20;20(7):494-507. Epub 2014 May 20.

Department of Chemical Sciences and Technologies, University of Rome 'Tor Vergata', I-00133, Rome, Italy.

Interactions between peptides are relevant from a biomedical point of view, in particular for the role played by their aggregates in different important pathologies, and also because peptide aggregates represent promising scaffolds for innovative materials. In the present article, the aggregation properties of the homo-peptides formed by α-aminoisobutyric acid (U) residues are discussed. The peptides investigated have chain lengths between six and 15 residues and comprise benzyl and naphthyl groups at the N- and C-termini, respectively. Spectroscopic experiments and molecular dynamics simulations show that the shortest homo-peptide, constituted by six U, does not exhibit any tendency to aggregate under the conditions examined. On the other hand, the homologous peptide with 15 U forms very stable and compact aggregates in 70/30(v/v) methanol/water solution. Atomic force microscopy images indicate that these aggregates promote formation of long fibrils once they are deposited on a mica surface. The aggregation phenomenon is mainly due to hydrophobic interactions occurring between very stable helical structures, and the aromatic groups in the peptides seem to play a minor role.
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http://dx.doi.org/10.1002/psc.2648DOI Listing
July 2014

Novel SMAD4 mutation causing Myhre syndrome.

Am J Med Genet A 2014 Jul 8;164A(7):1835-40. Epub 2014 Apr 8.

Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Rome, Italy.

Myhre syndrome (MYHRS, OMIM 139210) is an autosomal dominant disorder characterized by developmental and growth delay, athletic muscular built, variable cognitive deficits, skeletal anomalies, stiffness of joints, distinctive facial gestalt and deafness. Recently, SMAD4 (OMIM 600993) was identified by exome sequencing as the disease gene mutated in MYHRS. Previously only three missense mutations affecting Ile500 (p.Ile500Thr, p.Ile500Val, and p.Ile500Met) have been described in 22 unrelated subjects with MYHRS or a clinically related phenotype. Here we report on a 15-year-old boy with typical MYHRS and a novel heterozygous SMAD4 missense mutation affecting residue Arg496. This finding provides further information about the distinctive SMAD4 mutation spectrum in MYHRS. In silico structural analyses exploring the impact of the Arg-to-Cys change at codon 496 suggested that conformational changes promoted by replacement of Arg496 impact the stability of the SMAD heterotrimer and/or proper SMAD4 ubiquitination.
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http://dx.doi.org/10.1002/ajmg.a.36544DOI Listing
July 2014

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

Hum Mol Genet 2014 Aug 4;23(16):4315-27. Epub 2014 Apr 4.

Genetica Clinica Pediatrica, Clinica Pediatrica Università Milano Bicocca, Fondazione MBBM, A.O. S. Gerardo, Monza 20900, Italy.

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.
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http://dx.doi.org/10.1093/hmg/ddu148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103678PMC
August 2014

Rheoreversible hydrogels in paper restoration processes: a versatile tool.

Chem Cent J 2014 Feb 10;8(1):10. Epub 2014 Feb 10.

Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma "Tor Vergata", Via della Ricerca Scientifica snc, Rome 00133, Italy.

Background: Paper based artworks are probably ones of the most difficult materials to restore, because of their complexity and fragile structure. Cleaning of paper artifacts, one of the process commonly carried out during restoration, usually involves the use of solvents (organic or not), that may cause several troubles, like swelling and dissolution of some components, and may also be harmful to the users.

Results: Innovative procedure for cleaning paper artworks is reported in this paper. It is based on the use of rheoreversible, biocompatible hydrogels containing poly(ethylene oxide) or poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) and α-cyclodextrin. We have studied two types of polymer with different hydrophobic properties in order to obtain two different hydrogels with slightly different cleaning capabilities. Our overall strategy has been to develop innovative systems based on these hydrogels so as to better confront the problems that a restorer faces during the cleaning of paper samples. Rheoreversible hydrogels are intriguing materials because their application and removal is not invasive and does not require a liquid treatment that could induce damage to the paper.

Conclusions: These hydrogels have been applied in the cleaning of both new and aged paper samples and their cleaning efficiency has been established. Moreover, by comparison with traditional methods, the greater efficacy of the proposed procedure has been demonstrated.To assess the cleaning efficacy of these hydrogels, a multidisciplinary approach, combining non-invasive spectroscopic infrared techniques together with scanning electron microscopy, chromatographic (HPLC) analysis and pH investigations has been used. Near infrared spectroscopy spectra were coupled with a chemometric analysis to achieve a better interpretation of data.This work constitutes a preliminary step towards focused study in the development of α-cyclodextrin/polymer hydrogel family which will allow cleaning of paper artifacts with peculiar characteristics.
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http://dx.doi.org/10.1186/1752-153X-8-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926867PMC
February 2014

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

J Pept Sci 2013 Dec 31;19(12):758-69. Epub 2013 Oct 31.

Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy.

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.
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http://dx.doi.org/10.1002/psc.2574DOI Listing
December 2013

3D structure, dynamics, and activity of synthetic analog of the peptaibiotic trichodecenin I.

Chem Biodivers 2013 May;10(5):887-903

Department of Chemical Sciences and Technologies, University of Rome 'Tor Vergata', I-00133 Rome.

In this contribution, we report on the conformational preferences of synthetic analogs of the antimicrobial peptide trichodecenin I in solution. This 6-amino acid residue long peptide is characterized by a single, strongly helicogenic Aib residue in the central part of the sequence and is rich in the conformationally mobile Gly residues. It has been reported that, in CHCl3 solution and in the crystal state, this peptaibiotic adopts a non-helical, multiple β-turn conformation, whereas a 310 /α-helical structure was obtained from an X-ray diffraction study on a trichodecenin I analog (TDT4W6) containing the fluorescent Trp residue in position 6 (replacing Ile) and an equally helicogenic TOAC residue in position 4 (replacing Aib). In this work, we applied spectroscopic techniques and molecular-dynamics calculations, in particular, on the fluorescent TDT4W6 trichodecenin I analog with the aim at investigating its 3D-structural and dynamical features in solution. Our results revealed that TDT4W6 can be described by an ensemble of conformers quickly interconverting in the nanosecond time scale. The most populated cluster has a conformation similar to the NMR structure of native trichodecenin I in CHCl3 . However, also helical-like conformers are present, even if poorly populated and less stable under the analytical conditions.
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http://dx.doi.org/10.1002/cbdv.201200388DOI Listing
May 2013

Counteracting effects operating on Src homology 2 domain-containing protein-tyrosine phosphatase 2 (SHP2) function drive selection of the recurrent Y62D and Y63C substitutions in Noonan syndrome.

J Biol Chem 2012 Aug 18;287(32):27066-77. Epub 2012 Jun 18.

Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, 00161 Rome, Italy.

Activating mutations in PTPN11 cause Noonan syndrome, the most common nonchromosomal disorder affecting development and growth. PTPN11 encodes SHP2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase that positively modulates RAS function. Here, we characterized functionally all possible amino acid substitutions arising from single-base changes affecting codons 62 and 63 to explore the molecular mechanisms lying behind the largely invariant occurrence of the Y62D and Y63C substitutions recurring in Noonan syndrome. We provide structural and biochemical data indicating that the autoinhibitory interaction between the N-SH2 and protein-tyrosine phosphatase (PTP) domains is perturbed in both mutants as a result of an extensive structural rearrangement of the N-SH2 domain. Most mutations affecting Tyr(63) exerted an unpredicted disrupting effect on the structure of the N-SH2 phosphopeptide-binding cleft mediating the interaction of SHP2 with signaling partners. Among all the amino acid changes affecting that codon, the disease-causing mutation was the only substitution that perturbed the stability of the inactive conformation of SHP2 without severely impairing proper phosphopeptide binding of N-SH2. On the other hand, the disruptive effect of the Y62D change on the autoinhibited conformation of the protein was balanced, in part, by less efficient binding properties of the mutant. Overall, our data demonstrate that the selection-by-function mechanism acting as driving force for PTPN11 mutations affecting codons 62 and 63 implies balancing of counteracting effects operating on the allosteric control of the function of SHP2.
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http://dx.doi.org/10.1074/jbc.M112.350231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411048PMC
August 2012
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