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1.
Clin Genet ; 99(3): 457-461, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33354767

RESUMO

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.

2.
Am J Hum Genet ; 107(6): 1062-1077, 2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33217309

RESUMO

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-6Tyr553Cys, but not ClC-6WT, 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-6Tyr553Cys/Glu200Ala or ClC-6Glu200Ala 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.

3.
Soft Matter ; 16(44): 10169-10179, 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33165494

RESUMO

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 ß-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.

4.
Am J Hum Genet ; 107(3): 499-513, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32721402

RESUMO

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.


Assuntos
Carcinogênese/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Transtornos do Neurodesenvolvimento/genética , Síndrome de Noonan/genética , Pré-Escolar , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/patologia , Síndrome de Noonan/fisiopatologia , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais , Sequenciamento Completo do Exoma , Proteínas ras/genética
5.
J Chem Inf Model ; 60(6): 3157-3171, 2020 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-32395997

RESUMO

SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by PTPN11, plays a fundamental role in the modulation of several signaling pathways. Germline and somatic mutations in PTPN11 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 ß sheet and the loop that closes the pY binding pocket.

6.
Nanoscale Horiz ; 5(3): 553-563, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32118232

RESUMO

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.


Assuntos
Simulação de Dinâmica Molecular , Nanopartículas/química , Som , Transporte Biológico , Imagem Molecular , Nanopartículas/efeitos da radiação
7.
Parkinsonism Relat Disord ; 72: 75-79, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32120303

RESUMO

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.

8.
Hum Mutat ; 41(6): 1171-1182, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32112654

RESUMO

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 (Gln255 -to-Gln257 ). 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 Gln256 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 Gln257 variably enhance SHP2's catalytic activity, while missense changes involving Gln256 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.

9.
Front Chem ; 7: 170, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984741

RESUMO

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 Cα,α-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 in vitro at nanomolar concentrations and were resistant to proteolytic degradation.

10.
Am J Hum Genet ; 103(4): 621-630, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30290154

RESUMO

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.


Assuntos
Ativação do Canal Iônico/genética , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Canais de Potássio/genética , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Simulação de Dinâmica Molecular
11.
Hum Mutat ; 39(7): 959-964, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29737001

RESUMO

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.


Assuntos
Anormalidades Múltiplas/genética , Calcinose/genética , Otopatias/genética , Predisposição Genética para Doença , Deficiência Intelectual/genética , Atrofia Muscular/genética , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Anormalidades Múltiplas/fisiopatologia , Calcinose/fisiopatologia , Criança , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 3/genética , Hibridização Genômica Comparativa , Otopatias/fisiopatologia , Feminino , Humanos , Deficiência Intelectual/fisiopatologia , Masculino , Atrofia Muscular/fisiopatologia , Mutação de Sentido Incorreto/genética , Dedos de Zinco/genética
12.
Chem Commun (Camb) ; 54(39): 4943-4946, 2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29700513

RESUMO

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.


Assuntos
Antibacterianos/farmacologia , Peptidomiméticos/farmacologia , Tensoativos/farmacologia , Animais , Antibacterianos/química , Antibacterianos/toxicidade , Linhagem Celular Transformada , Escherichia coli/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Conformação Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Peptidomiméticos/química , Peptidomiméticos/toxicidade , Pseudomonas aeruginosa/efeitos dos fármacos , Tensoativos/química , Tensoativos/toxicidade
13.
Nanoscale ; 10(16): 7544-7555, 2018 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-29637964

RESUMO

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.


Assuntos
Nanoestruturas , Proteínas/análise , Dióxido de Silício , Adsorção , Laminina/análise , Propriedades de Superfície
14.
Adv Healthc Mater ; 6(23)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28945012

RESUMO

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.


Assuntos
Cetuximab , Sistemas de Liberação de Medicamentos/métodos , Receptores ErbB/antagonistas & inibidores , Ouro , Nanopartículas Metálicas/química , Peptídeos , Polietilenoglicóis , Células CACO-2 , Cetuximab/química , Cetuximab/farmacologia , Receptores ErbB/metabolismo , Ouro/química , Ouro/farmacologia , Humanos , Peptídeos/química , Peptídeos/farmacologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia
15.
Molecules ; 22(7)2017 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-28737669

RESUMO

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.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Peptídeos/química , Proteínas/química , Água/química , Lipopolissacarídeos/química
16.
Molecules ; 22(3)2017 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-28282944

RESUMO

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.


Assuntos
Boratos/química , Glucanos/química , Concentração de Íons de Hidrogênio , Dicroísmo Circular , Elasticidade , Hidrogéis/química , Ligação de Hidrogênio , Conformação Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Reologia , Comprimidos/química , Viscosidade
17.
Hum Mutat ; 38(4): 451-459, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28074573

RESUMO

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 Leu261 , Leu262 , and Arg265 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 Leu262 and Arg265 exerted by disruption of the N-SH2/PTP autoinhibitory interaction. Molecular dynamics simulations suggested a more complex behavior for changes affecting Leu261 , 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.


Assuntos
Predisposição Genética para Doença/genética , Mutação , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Síndrome de Noonan/patologia , Ligação Proteica , Domínios Proteicos , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Domínios de Homologia de src
18.
Oncotarget ; 7(15): 19982-96, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-26799588

RESUMO

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 ß-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.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Carnitina O-Palmitoiltransferase/genética , Regulação Neoplásica da Expressão Gênica , Apoptose , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina O-Palmitoiltransferase/metabolismo , Proliferação de Células , Epigênese Genética , Feminino , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Humanos , Simulação de Dinâmica Molecular , Oxirredução , Células Tumorais Cultivadas
19.
Hum Mutat ; 36(11): 1080-7, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26173643

RESUMO

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.


Assuntos
Estudos de Associação Genética , Mutação , Síndrome de Noonan/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Son Of Sevenless/genética , Adolescente , Adulto , Alelos , Substituição de Aminoácidos , Criança , Análise Mutacional de DNA , Exoma , Facies , Feminino , Genótipo , Humanos , Masculino , Modelos Moleculares , Síndrome de Noonan/diagnóstico , Fenótipo , Conformação Proteica , Proteínas Son Of Sevenless/química , Adulto Jovem
20.
Am J Hum Genet ; 96(5): 816-25, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25865493

RESUMO

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.


Assuntos
Catarata/genética , Surdez/genética , Quinase 3 da Glicogênio Sintase/genética , Deficiência Intelectual/genética , Proteínas Proto-Oncogênicas c-maf/genética , Catarata/patologia , Síndrome de Down/genética , Síndrome de Down/patologia , Humanos , Deficiência Intelectual/patologia , Mutação , Fenótipo , Fosforilação , Convulsões/genética , Convulsões/patologia
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