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1.
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.

2.
Animals (Basel) ; 10(11)2020 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-33238648

RESUMO

Phlorotannins have been reported to have positive effects on pig health, including improved gut health and digestibility. In this study, we investigate the effect of phenolics found in two brown seaweeds, Ascophyllum nodosum and Fucus serratus, on in vitro dry matter digestibility of seaweeds and commercial pig feed. Phlorotannin extracts and whole seaweeds were supplemented into pig feed to test their effect on digestibility. Solid-phase extraction was used to purify the phenolics to phlorotannins. The results showed a slight decrease in the digestibility of pig feed that was found to be significant when phlorotannin extracts were added from either seaweed. However, when whole A. nodosum was added to the pig feed, the effect on digestibility was less pronounced. Specifically, no significant difference in digestibility was observed at inclusion rates up to 5%, and thereafter results varied. A difference in digestibility was also observed in the same species at the same inclusion rate, collected from different seasons. This suggests that other compounds, e.g., polysaccharides, are having an effect on digestibility when whole seaweeds are supplemented to animal feed. This research has also highlighted the need to base supplementation on phenolic concentration as opposed to a standardised percentage inclusion of seaweeds to ensure that digestibility is not adversely affected.

3.
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
4.
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.

5.
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.

6.
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.

7.
J Colloid Interface Sci ; 562: 391-399, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-31864015

RESUMO

This manuscript presents a comparative study of the physico-chemical behaviour of sulfobetaine-type single and double zwitterions and zwitterionic salts, and structurally similar mono- and di-cationic tetraalkylammonium salts in aqueous solutions. The study includes experimental determination of the density and viscosity of highly diluted aqueous solutions with derivation of the Jones-Dole viscosity B-coefficient, partial molal volumes at infinite dilution, and hydration numbers. The study also examines the effects of addition of the salts on the surface tension of cationic and anionic surfactants, upper critical solution temperature of a non-ionic surfactant, solubility of amino acids, and stability of a protein. The experimental investigation was performed taking a broad bottom-up approach with the aim to elucidate the effect of molecular architecture and charge (two versus four) on the degree of surface hydration of a molecule, kosmotropicity, and interactions with charged and hydrophilic/hydrophobic surfaces - all-important characteristics which define ability of a functional group to resist protein attachment. The novel multicharged zwitterionic materials have exhibited superior qualities, thus paving the way to development of a new platform in design of hydrophilic and anti-fouling surfaces by employing the four-charge bearing molecular motifs.

8.
Biochim Biophys Acta Biomembr ; 1862(2): 183107, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678022

RESUMO

Host defense peptides selectively kill bacterial and cancer cells (including those that are drug-resistant) by perturbing the permeability of their membranes, without being significantly toxic to the host. Coulombic interactions between these cationic and amphipathic peptides and the negatively charged membranes of pathogenic cells contribute to the selective toxicity. However, a positive charge is not sufficient for selectivity, which can be achieved only by a finely tuned balance of electrostatic and hydrophobic driving forces. A common property of amphipathic peptides is the formation of aggregated structures in solution, but the role of this phenomenon in peptide activity and selectivity has received limited attention. Our data on the anticancer peptide killerFLIP demonstrate that aggregation strongly increases peptide selectivity, by reducing the effective peptide hydrophobicity and thus the affinity towards membranes composed of neutral lipids (like the outer layer of healthy eukaryotic cell membranes). Aggregation is therefore a useful tool to modulate the selectivity of membrane active peptides and peptidomimetics.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Multimerização Proteica , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/química , Ligação Proteica
9.
Pharmaceutics ; 11(8)2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31430958

RESUMO

Amorphous solid dispersion (ASD) is one of the most promising enabling formulations featuring significant water solubility and bioavailability enhancements for biopharmaceutical classification system (BCS) class II and IV drugs. An accurate thermodynamic understanding of the ASD should be established for the ease of development of stable formulation with desired product performances. In this study, we report a first experimental approach combined with classic Flory-Huggins (F-H) modelling to understand the performances of ASD across the entire temperature and drug composition range. At low temperature and drug loading, water (moisture) was induced into the system to increase the mobility and accelerate the amorphous drug-amorphous polymer phase separation (AAPS). The binodal line indicating the boundary between one phase and AAPS of felodipine, PVPK15 and water ternary system was successfully measured, and the corresponding F-H interaction parameters (χ) for FD-PVPK15 binary system were derived. By combining dissolution/melting depression with AAPS approach, the relationship between temperature and drug loading with χ (Φ, T) for FD-PVPK15 system was modelled across the entire range as χ = 1.72 - 852/T + 5.17·Φ - 7.85·Φ2. This empirical equation can provide better understanding and prediction for the miscibility and stability of drug-polymer ASD at all conditions.

10.
Cell Death Dis ; 10(8): 569, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358731

RESUMO

Neutrophils are key components of the innate arm of the immune system and represent the frontline of host defense against intruding pathogens. However, neutrophils can also cause damage to the host. Nanomaterials are being developed for a multitude of different purposes and these minute materials may find their way into the body through deliberate or inadvertent exposure; understanding nanomaterial interactions with the immune system is therefore of critical importance. However, whereas numerous studies have focused on macrophages, less attention is devoted to nanomaterial interactions with neutrophils, the most abundant leukocytes in the blood. We discuss the impact of engineered nanomaterials on neutrophils and how neutrophils, in turn, may digest certain carbon-based materials such as carbon nanotubes and graphene oxide. We also discuss the role of the corona of proteins adsorbed onto the surface of nanomaterials and whether nanomaterials are sensed as pathogens by cells of the immune system.


Assuntos
Nanotubos de Carbono/efeitos adversos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Transdução de Sinais/imunologia , Animais , Exossomos/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , Imunidade Inata , Inflamassomos/metabolismo , Inflamação/metabolismo , Camundongos , Coroa de Proteína/metabolismo
11.
Chembiochem ; 20(16): 2141-2150, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31125169

RESUMO

Trichogin GA IV is a short peptaibol with antimicrobial activity. This uncharged, but amphipathic, sequence is aligned at the membrane interface and undergoes a transition to an aggregated state that inserts more deeply into the membrane, an assembly that predominates at a peptide-to-lipid ratio (P/L) of 1:20. In this work, the natural trichogin sequence was prepared and reconstituted into oriented lipid bilayers. The 15 N NMR chemical shift is indicative of a well-defined alignment of the peptide parallel to the membrane surface at P/Ls of 1:120 and 1:20. When the P/L is increased to 1:8, an additional peptide topology is observed that is indicative of a heterogeneous orientation, with helix alignments ranging from around the magic angle to perfectly in-plane. The topological preference of the trichogin helix for an orientation parallel to the membrane surface was confirmed by attenuated total reflection FTIR spectroscopy. Furthermore, 19 F CODEX experiments were performed on a trichogin sequence with 19 F-Phe at position 10. The CODEX decay is in agreement with a tetrameric complex, in which the 19 F sites are about 9-9.5 Šapart. Thus, a model emerges in which the monomeric peptide aligns along the membrane surface. When the peptide concentration increases, first dimeric and then tetrameric assemblies form, made up from helices oriented predominantly parallel to the membrane surface. The formation of these aggregates correlates with the release of vesicle contents including relatively large molecules.


Assuntos
Bicamadas Lipídicas/química , Lipopeptídeos/química , Fosfolipídeos/química , Sequência de Aminoácidos , Modelos Moleculares , Estrutura Molecular , Propriedades de Superfície
12.
Chembiochem ; 20(16): 2125-2132, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31095838

RESUMO

Trichogin is a natural peptide endowed with antimicrobial and antitumor activity. A member of the peptaibol family, trichogin possesses a C-terminal amino alcohol. In the past, this moiety was substituted for a methyl ester for synthetic purposes and it was observed that this apparently slight modification caused significant changes in the peptide bioactivity. With the aim of understanding the reasons behind such observations, a detailed spectroscopic study on a number of trichogin analogues has been performed. Herein, data obtained from synchrotron radiation circular dichroism, NMR spectroscopy, and fluorescence spectroscopy in organic solvents at cryogenic temperatures are compared with those independently acquired by means of EPR spectroscopy at 80 K. It is unambiguously revealed that the presence of a reversible, temperature-driven, screw-sense interconversion from a right- to left-handed helix is determined by the C-terminal capping moiety. Data demonstrate, for the first time, the key role of a C-terminal methyl ester in promoting peptide screw-sense inversion.


Assuntos
Peptaibols/química , Temperatura , Sequência de Aminoácidos , Amino Álcoois/química , Ácidos Carboxílicos/química , Ésteres/química , Conformação Proteica em alfa-Hélice , Relação Estrutura-Atividade
13.
Bioconjug Chem ; 30(7): 1998-2010, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31145591

RESUMO

A synthetic antimicrobial peptide library based on the human autophagy 16 polypeptide has been developed. Designed acetylated peptides bearing lipids of different chain lengths resulted in peptides with enhanced potency compared to the parent Atg16. A 21-residue fragment of Atg16 conjugated to 4-methylhexanoic acid (K30) emerged as the most potent antibacterial, with negligible hemolysis. Several studies, including microscopy, dye leakage, and ITC, were conducted to gain insight into the antibacterial mechanism of action of the peptide. Visual inspection using both SEM and TEM revealed the membranolytic effect of the peptide on bacterial cells. The selectivity of the peptide against bacterial cell membranes was also proven using dye leakage assays. ITC analysis revealed the exothermic nature of the binding interaction of the peptide to D8PG micelles. The three-dimensional solution NMR structure of K30 in complex with dioctanoylphosphatidylglycerol (D8PG) micelles revealed that the peptide adopts a helix-loop-helix structure in the presence of anionic membrane lipids mimicking bacterial membranes. Intermolecular NOEs between the peptide and lipid deciphered the location of the peptide in the bound state, which was subsequently supported by the paramagnetic relaxation enhancement (PRE) NMR experiment. Collectively, these results describe the structure-function relationship of the peptide in the bacterial membrane.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/farmacologia , Acilação , Sequência de Aminoácidos , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos
14.
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.

15.
Adv Exp Med Biol ; 1117: 175-214, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980359

RESUMO

Antimicrobial peptides (AMPs) attack bacterial membranes selectively, killing microbes at concentrations that cause no toxicity to the host cells. This selectivity is not due to interaction with specific receptors but is determined by the different lipid compositions of the membranes of the two cell types and by the peculiar physicochemical properties of AMPs, particularly their cationic and amphipathic character. However, the available data, including recent studies of peptide-cell association, indicate that this picture is excessively simplistic, because selectivity is modulated by a complex interplay of several interconnected phenomena. For instance, conformational transitions and self-assembly equilibria modulate the effective peptide hydrophobicity, the electrostatic and hydrophobic contributions to the membrane-binding driving force are nonadditive, and kinetic processes can play an important role in selective bacterial killing in the presence of host cells. All these phenomena and their bearing on the final activity and toxicity of AMPs must be considered in the definition of design principles to optimize peptide selectivity.


Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Bactérias , Membrana Celular/química , Lipídeos de Membrana/química , Testes de Sensibilidade Microbiana
16.
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
17.
FEBS J ; 285(17): 3225-3237, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30028086

RESUMO

Human serum albumin (HSA) is characterized by 17 disulfides and by only one unpaired cysteine (Cys34 ), which can be free in the reduced albumin or linked as a mixed disulfide with cysteine, or in minor amount with other natural thiols, in the oxidized albumin. In healthy subjects, the level of the oxidized form is about 35%, but it rises up to 70% after oxidative insults or in patients with kidney diseases. Oxidized albumin is therefore considered a short-term biomarker of oxidative stress as its level may increase or decrease under appropriate redox inputs in discrete temporal spans. This paper defines, for the first time, the kinetic properties of reduced and oxidized Cys34 of HSA in their reactions with natural disulfides and thiols. Kinetic constants support the evidence that the Cys34 redox oscillations observed in vivo are mainly due to the interaction with cysteine and cystine without the involvement of any enzymatic support. This study gives also a plausible explanation for the absence of involvement of the 17 disulfides naturally present in HSA in these redox transitions. This inert behavior toward cysteine is marginally due to solvent accessibility or flexibility factors of these bonds but mainly to their strong thermodynamic stability, which is caused essentially by a proximity effect. A similar mechanism is likely at play in the many proteins that maintain disulfide bridges in a reducing medium like the cytosol.


Assuntos
Biomarcadores/sangue , Cisteína/metabolismo , Dissulfetos/metabolismo , Diálise Renal , Albumina Sérica Humana/metabolismo , Compostos de Sulfidrila/metabolismo , Uremia/sangue , Estudos de Casos e Controles , Cisteína/química , Dissulfetos/química , Humanos , Oxirredução , Estresse Oxidativo , Albumina Sérica Humana/química , Compostos de Sulfidrila/química , Uremia/patologia
18.
Colloids Surf B Biointerfaces ; 168: 2-9, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29728291

RESUMO

Gold nanoparticles (AuNPs) are considered suitable systems for drug delivery and diagnostics with several applications in biomedicine. Size, shape and surface functionalization of these nanoparticles are important parameters influencing their behavior in a biological environment. This study describes the preparation and the characterization of lysophosphocholine coated AuNPs by means of Small Angle Neutron Scattering (SANS), Electron Paramagnetic Resonance (EPR) and Fluorescence Spectroscopy. In particular the structure of the functionalized AuNP suspension, as well as the physical properties, of the nanoparticle organic coating are discussed. The experimental results indicated that functionalized lysophosphocholine-AuNPs form aggregates, which are composed by nanoparticles with core-shell structure. Nevertheless, the nanoparticle suspension resulted to be stable, without significant structural rearrangements even when the temperature was increased to 50 °C. At the same time, experimental evidences also suggested that the 18LPC layer around AuNPs presented a reduced chain packing compared to pure 18LPC aggregates.


Assuntos
Materiais Revestidos Biocompatíveis/química , Ouro/química , Lipídeos/química , Nanopartículas Metálicas/química , Materiais Revestidos Biocompatíveis/síntese química , Espectroscopia de Ressonância de Spin Eletrônica , Lisofosfatidilcolinas/química , Espalhamento a Baixo Ângulo , Espectrometria de Fluorescência , Temperatura
19.
Sensors (Basel) ; 18(5)2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29747464

RESUMO

A novel type of graphene-like nanoparticle, synthesized by oxidation and unfolding of C60 buckminsterfullerene fullerene, showed multiple and reproducible sensitivity to Cu2+, Pb2+, Cd2+, and As(III) through different degrees of fluorescence quenching or, in the case of Cd2+, through a remarkable fluorescence enhancement. Most importantly, only for Cu2+ and Pb2+, the fluorescence intensity variations came with distinct modifications of the optical absorption spectrum. Time-resolved fluorescence study confirmed that the common origin of these diverse behaviors lies in complexation of the metal ions by fullerene-derived carbon layers, even though further studies are required for a complete explanation of the involved processes. Nonetheless, the different response of fluorescence and optical absorbance towards distinct cationic species makes it possible to discriminate between the presence of Cu2+, Pb2+, Cd2+, and As(III), through two simple optical measurements. To this end, the use of a three-dimensional calibration plot is discussed. This property makes fullerene-derived nanoparticles a promising material in view of the implementation of a selective, colorimetric/fluorescent detection system.

20.
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
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