Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 140
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Antioxidants (Basel) ; 11(4)2022 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-35453350

RESUMO

Hyaluronic acid (Hy) is a natural linear polymer that is widely distributed in different organisms, especially in the articular cartilage and the synovial fluid. During tissue injury due to oxidative stress, Hy plays an important protective role. All the beneficial properties of Hy make the polymer attractive for many biomedical uses; however, the low stability and short biological half-life limit Hy application. To overcome these problems, the addition of small antioxidant molecules to Hy solution has been employed to protect the molecular integrity of Hy or delay its degradation. Carnosine (ß-alanyl-L-histidine, Car) protects cells from the damage due to the reactive species derived from oxygen (ROS), nitrogen (RNS) or carbonyl groups (RCS). Car inhibits the degradation of hyaluronan induced by free radical processes in vitro but, like Hy, the potential protective action of Car is drastically hampered by the enzymatic hydrolysis in vivo. Recently, we conjugated Hy to Car and the derivatives (HyCar) showed protective effects in experimental models of osteoarthritis and rheumatoid arthritis in vivo. Here we report the antioxidant activity exerted by HyCar against ROS, RNS and RCS. Moreover, we tested if the covalent conjugation between Hy and Car inhibits the enzymatic hydrolysis of the polymer and the dipeptide backbone. We found that the antioxidant properties and the resistance to the enzymatic hydrolysis of Hy and Car are greatly improved by the conjugation.

2.
Int J Mol Sci ; 23(6)2022 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-35328348

RESUMO

Ctr1 regulates copper uptake and its intracellular distribution. The first 14 amino acid sequence of the Ctr1 ectodomain Ctr1(1-14) encompasses the characteristic Amino Terminal Cu2+ and Ni2+ binding motif (ATCUN) as well as the bis-His binding motif (His5 and His6). We report a combined thermodynamic and spectroscopic (UV-vis, CD, EPR) study dealing with the formation of Cu2+ homobinuclear complexes with Ctr1(1-14), the percentage of which is not negligible even in the presence of a small Cu2+ excess and clearly prevails at a M/L ratio of 1.9. Ascorbate fails to reduce Cu2+ when bound to the ATCUN motif, while it reduces Cu2+ when bound to the His5-His6 motif involved in the formation of binuclear species. The histidine diade characterizes the second binding site and is thought to be responsible for ascorbate oxidation. Binding constants and speciation of Ag+ complexes with Ctr1(1-14), which are assumed to mimic Cu+ interaction with N-terminus of Ctr1(1-14), were also determined. A preliminary immunoblot assay evidences that the anti-Ctr1 extracellular antibody recognizes Ctr1(1-14) in a different way from the longer Ctr1(1-25) that encompasses a second His and Met rich domain.


Assuntos
Proteínas de Transporte de Cátions , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte de Cátions/metabolismo , Cobre/química , Histidina/química
3.
Int J Mol Sci ; 22(24)2021 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-34948299

RESUMO

l-carnosine (ß-alanyl-l-histidine) (Car hereafter) is a natural dipeptide widely distributed in mammalian tissues and reaching high concentrations (0.7-2.0 mM) in the brain. The molecular features of the dipeptide underlie the antioxidant, anti-aggregating and metal chelating ability showed in a large number of physiological effects, while the biological mechanisms involved in the protective role found against several diseases cannot be explained on the basis of the above-mentioned properties alone, requiring further research efforts. It has been reported that l-carnosine increases the secretion and expression of various neurotrophic factors and affects copper homeostasis in nervous cells inducing Cu cellular uptake in keeping with the key metal-sensing system. Having in mind this l-carnosine ability, here we report the copper-binding and ionophore ability of l-carnosine to activate tyrosine kinase cascade pathways in PC12 cells and stimulate the expression of BDNF. Furthermore, the study was extended to verify the ability of the dipeptide to favor copper signaling inducing the expression of VEGF. Being aware that the potential protective action of l-carnosine is drastically hampered by its hydrolysis, we also report on the behavior of a conjugate of l-carnosine with trehalose that blocks the carnosinase degradative activity. Overall, our findings describe a copper tuning effect on the ability of l-carnosine and, particularly its conjugate, to activate tyrosine kinase cascade pathways.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Carnosina/farmacologia , Cobre/metabolismo , Ionóforos/farmacologia , Trealose/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Quelantes/farmacologia , Dipeptídeos/metabolismo , Células PC12 , Ratos , Transdução de Sinais
4.
Org Biomol Chem ; 19(43): 9427-9432, 2021 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-34668911

RESUMO

Trehalose, a non-reducing disaccharide of glucose, is a natural bioactive and non-toxic sugar. It is found in many organisms that synthesise it when their cells are exposed to stress conditions. While not produced by mammalian cells, this disaccharide and also some of its derivatives have been shown to have a number of interesting properties that indicate their importance in the treatment of certain human diseases. Differentiating the two glucosyl moieties in the trehalose molecule has often been a synthetic challenge. We report here an easy way to obtain the monoaldehyde of trehalose, as well as the relevant symmetrical dialdehyde. The reactivity of the aldehyde functionalities involved in the molecular structure of these synthons allows the easy preparation of the corresponding amino or carboxy derivatives of trehalose, as well the synthesis of some new trehalose conjugates useful for diagnostic or therapeutic purposes.


Assuntos
Aldeídos/química , Trealose/química , Animais , Humanos , Estrutura Molecular , Oxirredução
5.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064906

RESUMO

Nerve growth factor (NGF) is a protein essential to neurons survival, which interacts with its receptor as a non-covalent dimer. Peptides belonging to NGF N-terminal domain are able to mimic the activity of the whole protein. Such activity is affected by the presence of copper ions. The metal is released in the synaptic cleft where proteins, not yet identified, may bind and transfer to human copper transporter 1 (hCtr1), for copper uptake in neurons. The measurements of the stability constants of copper complexes formed by amyloid beta and hCtr1 peptide fragments suggest that beta-amyloid (Aß) can perform this task. In this work, the stability constant values of copper complex species formed with the dimeric form of N-terminal domain, sequence 1-15 of the protein, were determined by means of potentiometric measurements. At physiological pH, NGF peptides bind one equivalent of copper ion with higher affinity of Aß and lower than hCtr1 peptide fragments. Therefore, in the synaptic cleft, NGF may act as a potential copper chelating molecule, ionophore or chaperone for hCtr1 for metal uptake. Copper dyshomeostasis and mild acidic environment may modify the balance between metal, NGF, and Aß, with consequences on the metal cellular uptake and therefore be among causes of the Alzheimer's disease onset.


Assuntos
Transportador de Cobre 1/metabolismo , Cobre/metabolismo , Fator de Crescimento Neural/metabolismo , Fragmentos de Peptídeos/metabolismo , Sítios de Ligação , Humanos , Ligação Proteica
6.
Int J Mol Sci ; 21(20)2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-33066163

RESUMO

We investigate the interaction of hemin with four fragments of prion protein (PrP) containing from one to four histidines (PrP106-114, PrP95-114, PrP84-114, PrP76-114) for its potential relevance to prion diseases and possibly traumatic brain injury. The binding properties of hemin-PrP complexes have been evaluated by UV-visible spectrophotometric titration. PrP peptides form a 1:1 adduct with hemin with affinity that increases with the number of histidines and length of the peptide; the following log K1 binding constants have been calculated: 6.48 for PrP76-114, 6.1 for PrP84-114, 4.80 for PrP95-114, whereas for PrP106-114, the interaction is too weak to allow a reliable binding constant calculation. These constants are similar to that of amyloid-ß (Aß) for hemin, and similarly to hemin-Aß, PrP peptides tend to form a six-coordinated low-spin complex. However, the concomitant aggregation of PrP induced by hemin prevents calculation of the K2 binding constant. The turbidimetry analysis of [hemin-PrP76-114] shows that, once aggregated, this complex is scarcely soluble and undergoes precipitation. Finally, a detailed study of the peroxidase-like activity of [hemin-(PrP)] shows a moderate increase of the reactivity with respect to free hemin, but considering the activity over long time, as for neurodegenerative pathologies, it might contribute to neuronal oxidative stress.


Assuntos
Hemina/química , Fragmentos de Peptídeos/química , Proteínas Priônicas/química , Sítios de Ligação , Oxirredução , Fragmentos de Peptídeos/metabolismo , Polimerização , Ligação Proteica
7.
Sci Rep ; 10(1): 15998, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994475

RESUMO

Alzheimer's disease is the most common neurodegenerative disorder. Finding a pharmacological approach that cures and/or prevents the onset of this devastating disease represents an important challenge for researchers. According to the amyloid cascade hypothesis, increases in extracellular amyloid-ß (Aß) levels give rise to different aggregated species, such as protofibrils, fibrils and oligomers, with oligomers being the more toxic species for cells. Many efforts have recently been focused on multi-target ligands to address the multiple events that occur concurrently with toxic aggregation at the onset of the disease. Moreover, investigating the effect of endogenous compounds or a combination thereof is a promising approach to prevent the side effects of entirely synthetic drugs. In this work, we report the synthesis, structural characterization and Aß antiaggregant ability of new derivatives of hyaluronic acid (Hy, 200 and 700 kDa) functionalized with carnosine (Car), a multi-functional natural dipeptide. The bioactive substances (HyCar) inhibit the formation of amyloid-type aggregates of Aß42 more than the parent compounds; this effect is proportional to Car loading. Furthermore, the HyCar derivatives are able to dissolve the amyloid fibrils and to reduce Aß-induced toxicity in vitro. The enzymatic degradation of Aß is also affected by the interaction with HyCar.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Carnosina/farmacologia , Ácido Hialurônico/farmacologia , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/efeitos dos fármacos , Carnosina/química , Linhagem Celular , Humanos , Ácido Hialurônico/química , Modelos Biológicos , Estrutura Molecular , Agregados Proteicos/efeitos dos fármacos
8.
ACS Omega ; 5(29): 17900-17907, 2020 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-32743161

RESUMO

Metallothioneins (MTs) are metal-binding proteins that are overexpressed in various human cancers and are thought to be associated with resistance to cytotoxic drugs. The knowledge on MT expression, regulation, and function in human gliomas is limited. We found that MT3 mRNA was highly expressed in cell lines derived from grade IV gliomas (i.e., A172 and U87 cells), as compared to grade II astrocytoma cells (i.e., 1321N1). Different from 1321N1, U87 cells were partly resistant to the alkylating drug, temozolomide (TMZ) (100 µM for 96 h), which induced a massive accumulation of U87 into the S and G2 fractions of the cell cycle but not apoptotic death. Silencing of MT3 did not significantly affect U87 cell proliferation and survival, but it delayed G1/S transition and favored the occurrence of apoptosis in TMZ-treated cells. Accordingly, the combination of MT3 silencing and TMZ treatment increased the protein levels of checkpoint kinase-1, which was ultimately responsible for the lasting G1 arrest and death of double treated U87 cells.

9.
Chemistry ; 26(57): 13072-13084, 2020 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-32488947

RESUMO

Islet amyloid polypeptide (IAPP) is a hormone co-secreted with insulin and zinc from pancreatic ß-cells. To overcome the low solubility of human IAPP, we characterized zinc complexes species formed with 1) a mutated form of rat-IAPP(1-37; R18 H) able to mimic the human IAPP, 2) the r-IAPP(1-37) and the IAPP(1-8) fragment. Stoichiometry, speciation and coordination features of zinc(II) complexes were unveiled by ESI-MS, potentiometry and NMR measurements combined with DFT and free-energy simulations. Mononuclear species start to form around pH 6; Zn2+ binds both His18 and N-amino terminus in rat-IAPP(1-37; R18 H). The in silico study allows us to assess not only a structured turn compact domain in r-IAPP(1-37) and r-IAPP(1-37; R18 H) featured by a different free energy barrier for the transition from the compact to elongated conformation upon the coordination of Zn2+ , but also to bring into light a coordination shell further stabilized by noncovalent interactions.


Assuntos
Zinco/química , Amiloide , Animais , Simulação por Computador , Complexos de Coordenação , Humanos , Insulina , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Ratos
10.
Int J Mol Sci ; 21(10)2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32408629

RESUMO

Thyroid cancer incidence is significantly increased in volcanic areas, where relevant non-anthropogenic pollution with heavy metals is present in the environment. This review will discuss whether chronic lifelong exposure to slightly increased levels of metals can contribute to the increase in thyroid cancer in the residents of a volcanic area. The influence of metals on living cells depends on the physicochemical properties of the metals and their interaction with the target cell metallostasis network, which includes transporters, intracellular binding proteins, and metal-responsive elements. Very little is known about the carcinogenic potential of slightly increased metal levels on the thyroid, which might be more sensitive to mutagenic damage because of its unique biology related to iodine, which is a very reactive and strongly oxidizing agent. Different mechanisms could explain the specific carcinogenic effect of borderline/high environmental levels of metals on the thyroid, including (a) hormesis, the nonlinear response to chemicals causing important biological effects at low concentrations; (b) metal accumulation in the thyroid relative to other tissues; and (c) the specific effects of a mixture of different metals. Recent evidence related to all of these mechanisms is now available, and the data are compatible with a cause-effect relationship between increased metal levels in the environment and an increase in thyroid cancer incidence.


Assuntos
Poluição Ambiental/efeitos adversos , Metais Pesados/análise , Neoplasias da Glândula Tireoide/etiologia , Erupções Vulcânicas/efeitos adversos , Exposição Ambiental/efeitos adversos , Monitoramento Ambiental/métodos , Humanos , Incidência , Neoplasias da Glândula Tireoide/epidemiologia
11.
Biomed Pharmacother ; 125: 110023, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32092830

RESUMO

Several studies demonstrated the pharmacological actions of carnosine as well as hyaluronic acid (HA) during joint inflammation. In that regard, the aim of this study was to investigate the protective effect of a new HA -Carnosine conjugate (FidHycarn) on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA). CIA was induced by two intradermal injections of 100 µl of an emulsion of collagen (CII) and complete Freund's adjuvant (CFA) at the base of the tail on day 0 and 21. At 35 day post CIA induction, the animals were sacrificed. CII injection caused erythema and edema in the hind paws, histological alterations with erosion of the joint cartilage as well as behavioral changes. Oral treatment with FidHycarn starting at the onset of arthritis (day 25) ameliorated the clinical signs, improved behavioral deficits as well as decreased histological and radiographic alterations. The degree of oxidative damage evaluated by inducible nitric oxide synthase (iNOS), nitrotyrosine, poly-ADP-ribose (PAR) expressions and malondialdehyde (MDA) levels, was also significantly reduced in Carnosine+HA association and FidHycarn treated mice. Moreover, the levels of proinflammatory cytokines and chemokines and cyclo-oxygenase COX-2 enzyme were also more significantly reduced by Carnosine+HA and FidHycarn compared to carnosine alone. However, interestingly, in some cases, the effects of FidHycarn were more important than Carnosine+HA association and not statistically different to methotrexate (MTX) used as positive control. Thus, the conjugation of Carnosine with HA (FidHycarn) could represent an interesting therapeutic strategy to combat arthritis disorders.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Carnosina , Ácido Hialurônico , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Animais , Anti-Inflamatórios/síntese química , Artrite Experimental/diagnóstico por imagem , Artrite Experimental/etiologia , Artrite Experimental/patologia , Biomarcadores , Carnosina/química , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Ácido Hialurônico/química , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Oxirredução , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Substâncias Protetoras/síntese química , Radiografia
12.
Biomolecules ; 10(2)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32024191

RESUMO

In the last decade, Nerve Growth Factor (NGF)-based clinical approaches have lacked specific and efficient Tyrosine Kinase A (TrkA) agonists for brain delivery. Nowadays, the characterization of novel small peptidomimetic is taking centre stage in preclinical studies, in order to overcome the main size-related limitation in brain delivery of NGF holoprotein for Central Nervous System (CNS) pathologies. Here we investigated the NGF mimetic properties of the human NGF 1-14 sequence (hNGF1-14) and its derivatives, by resorting to primary cholinergic and dorsal root ganglia (DRG) neurons. Briefly, we observed that: 1) hNGF1-14 peptides engage the NGF pathway through TrkA phosphorylation at tyrosine 490 (Y490), and activation of ShcC/PI3K and Plc-γ/MAPK signalling, promoting AKT-dependent survival and CREB-driven neuronal activity, as seen by levels of the immediate early gene c-Fos, of the cholinergic marker Choline Acetyltransferase (ChAT), and of Brain Derived Neurotrophic Factor (BDNF); 2) their NGF mimetic activity is lost upon selective TrkA inhibition by means of GW441756; 3) hNGF1-14 peptides are able to sustain DRG survival and differentiation in absence of NGF. Furthermore, the acetylated derivative Ac-hNGF1-14 demonstrated an optimal NGF mimetic activity in both neuronal paradigms and an electrophysiological profile similar to NGF in cholinergic neurons. Cumulatively, the findings here reported pinpoint the hNGF1-14 peptide, and in particular its acetylated derivative, as novel, specific and low molecular weight TrkA specific agonists in both CNS and PNS primary neurons.


Assuntos
Neurônios Colinérgicos/metabolismo , Gânglios Espinais/metabolismo , Fator de Crescimento Neural/química , Receptor trkA/agonistas , Receptor trkA/metabolismo , Proteína 3 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Animais , Bioensaio , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Humanos , Peptídeos/química , Fosforilação , Ratos , Transdução de Sinais , Tirosina/química
13.
Inorg Chem ; 59(1): 900-912, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31869218

RESUMO

The combination between dyshomeostatic levels of catecholamine neurotransmitters and redox-active metals such as copper and iron exacerbates the oxidative stress condition that typically affects neurodegenerative diseases. We report a comparative study of the oxidative reactivity of copper complexes with amyloid-ß (Aß40) and the prion peptide fragment 76-114 (PrP76-114), containing the high-affinity binding site, toward dopamine and 4-methylcatechol, in aqueous buffer and in sodium dodecyl sulfate micelles, as a model membrane environment. The competitive oxidative and covalent modifications undergone by the peptides were also evaluated. The high binding affinity of Cu/peptide to micelles and lipid membranes leads to a strong reduction (Aß40) and quenching (PrP76-114) of the oxidative efficiency of the binary complexes and to a stabilization and redox silencing of the ternary complex CuII/Aß40/PrP76-114, which is highly reactive in solution. The results improve our understanding of the pathological and protective effects associated with these complexes, depending on the physiological environment.


Assuntos
Peptídeos beta-Amiloides/química , Cobre/química , Dopamina/química , Príons/química , Dodecilsulfato de Sódio/química , Sítios de Ligação , Humanos , Micelas , Conformação Molecular , Solubilidade
14.
Metallomics ; 11(9): 1567-1578, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31482903

RESUMO

In this work we report on the synthesis and physiochemical/biological characterization of a peptide encompassing the first thirteen residues of neurotrophin-3 (NT-3). The protein capability to promote neurite outgrowth and axonal branching by a downstream mechanism that involves the increase of the cAMP response element-binding level (CREB) was found for the NT3(1-13) peptide, thus validating its protein mimetic behaviour. Since copper ions are also involved in neurotransmission and their internalization may be an essential step in neuron differentiation and CREB phosphorylation, the peptide and its copper complexes were characterized by potentiometric and spectroscopic techniques, including UV-visible, CD and EPR. To have a detailed picture of the coordination features of the copper complexes with NT3(1-13), we also scrutinized the two peptide fragments encompassing the shorter sequences 1-5 and 5-13, respectively, showing that the amino group is the main anchoring site for Cu(ii) at physiological pH. The peptide activity increased in the presence of copper ions. The effect of copper(ii) addition is more marked for NT3(1-13) than the other two peptide fragments, in agreement with its higher affinity for metal ions. Confocal microscopy measurements carried out on fluorescently labelled NT3(1-13) indicated that copper ions increase peptide internalization.


Assuntos
Complexos de Coordenação/farmacologia , Cobre/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Neurotrofina 3/farmacologia , Peptídeos/farmacologia , Linhagem Celular , Complexos de Coordenação/química , Cobre/química , Humanos , Neurotrofina 3/química , Peptídeos/química , Fosforilação/efeitos dos fármacos
15.
Molecules ; 24(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370315

RESUMO

Conflicting values, obtained by different techniques and often under different experimental conditions have been reported on the affinity of Zn2+ for amyloid-ß, that is recognized as the major interaction responsible for Alzheimer's disease. Here, we compare the approaches employed so far, i.e., the evaluation of Kd and the determination of the stability constants to quantitatively express the affinity of Zn2+ for the amyloid-ß peptide, evidencing the pros and cons of the two approaches. We also comment on the different techniques and conditions employed that may lead to divergent data. Through the analysis of the species distribution obtained for two selected examples, we show the implications that the speciation, based on stoichiometric constants rather than on Kd, may have on data interpretation. The paper also demonstrates that the problem is further complicated by the occurrence of multiple equilibria over a relatively narrow pH range.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Zinco/química , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Fragmentos de Peptídeos/genética , Ligação Proteica/genética
16.
J Inorg Biochem ; 199: 110759, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31299377

RESUMO

Copper is involved in several biological processes. The static and labile copper pools are controlled by means of a network of influx and efflux transporters, storage proteins, chaperones, transcription factors and small molecules as glutathione (GSH), which contributes to the cell reducing environment. To follow the fate of intracellular copper labile pool, a variant of human apocarbonic anhydrase has been proposed as fluorescent probe to monitor cytoplasmic Cu2+. Aware that in this cellular compartment copper ion is present as Cu+, electron spin resonance technique (ESR) was used to ascertain whether (bovine or human) carbonic anhydrase (CA) was able to accommodate Cu+ in the same sites occupied by Cu2+, in the presence of naturally occurring reducing agents such as ascorbate and GSH. Our ESR results on Cu2+ complexes with CA allow for a complete characterization of the two metal binding sites of the protein in solution. The use of the reported affinity constants of zinc in the catalytic site and of Cu2+ in the peripheral and catalytic site, allow us to obtain the speciation of copper species mimicking the spectroscopic study conditions. The different Cu2+ coordination features in the catalytic and the peripheral (the N-terminus cleft mouth) binding sites influence the chemical reduction effect of the two main naturally occurring reductants. Ascorbate reversibly reduces the Cu2+ complex with CA, while glutathione irreversibly induces the formation of Cu2+ complex with its oxidized form (GSSG). Our results questioned the use of CA as intracellular Cu2+ sensor. Furthermore, translating these findings to intracellular environment, the conversion of GSH in GSSG can significantly alter the metallostasis.


Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Cobre/química , Substâncias Redutoras/metabolismo , Animais , Ácido Ascórbico/metabolismo , Sítios de Ligação , Catálise , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Glutationa/metabolismo , Humanos , Oxirredução , Oxigênio/metabolismo , Ligação Proteica
17.
Cells ; 8(4)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939824

RESUMO

Nerve growth factor (NGF) is a protein necessary for development and maintenance of the sympathetic and sensory nervous systems. We have previously shown that the NGF N-terminus peptide NGF(1-14) is sufficient to activate TrkA signaling pathways essential for neuronal survival and to induce an increase in brain-derived neurotrophic factor (BDNF) expression. Cu2+ ions played a critical role in the modulation of the biological activity of NGF(1-14). Using computational, spectroscopic, and biochemical techniques, here we report on the ability of a newly synthesized peptide named d-NGF(1-15), which is the dimeric form of NGF(1-14), to interact with TrkA. We found that d-NGF(1-15) interacts with the TrkA-D5 domain and induces the activation of its signaling pathways. Copper binding to d-NGF(1-15) stabilizes the secondary structure of the peptides, suggesting a strengthening of the noncovalent interactions that allow for the molecular recognition of D5 domain of TrkA and the activation of the signaling pathways. Intriguingly, the signaling cascade induced by the NGF peptides ultimately involves cAMP response element-binding protein (CREB) activation and an increase in BDNF protein level, in keeping with our previous result showing an increase of BDNF mRNA. All these promising connections can pave the way for developing interesting novel drugs for neurodegenerative diseases.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cobre/farmacologia , Fator de Crescimento Neural/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dimerização , Endocitose/efeitos dos fármacos , Feminino , Ionóforos/farmacologia , Fator de Crescimento Neural/química , Células PC12 , Fenótipo , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Ratos , Ratos Wistar , Receptor trkA/química , Receptor trkA/metabolismo , Termodinâmica
18.
Oncotarget ; 9(91): 36289-36316, 2018 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-30555630

RESUMO

Copper homeostasis is generally investigated focusing on a single component of the metallostasis network. Here we address several of the factors controlling the metallostasis for neuroblastoma cells (SH-SY5Y) upon treatment with 2,9-dimethyl-1,10-phenanthroline-5,6-dione (phendione) and 2,9-dimethyl-1,10-phenanthroline (cuproindione). These compounds bind and transport copper inside cells, exert their cytotoxic activity through the induction of oxidative stress, causing apoptosis and alteration of the cellular redox and copper homeostasis network. The intracellular pathway ensured by copper transporters (Ctr1, ATP7A), chaperones (CCS, ATOX, COX 17, Sco1, Sco2), small molecules (GSH) and transcription factors (p53) is scrutinised.

20.
J Cell Biochem ; 119(12): 9707-9719, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30129075

RESUMO

Zinc is a transition metal and catalytic cofactor involved in many biological processes including proliferation, development, differentiation, and metabolism. Zinc transporters (ZnTs) play a fundamental role in cellular zinc homeostasis. ZnTs are responsible of zinc efflux and are encoded by 10 genes belonging to solute carrier family 30A (SLC30A1-10), while zinc-regulated transporter (ZRT)/iron-regulated transporter (IRT)-like protein (ZIP) transporters are responsible for the influx of zinc into the cytoplasm and are encoded by 14 genes belonging to solute carrier family 39A (SLC39A1-14). In this study, we analyzed, by transcriptome analysis, the microRNA levels of ZnT-encoding and ZIP-encoding genes in colorectal cancer (CRC) samples matched to normal colon tissues and in CRC cell lines. Results revealed an upregulation of specific ZnT and ZIP transcripts in CRC. Upregulation of SLC30A5, SLC30A6, SLC30A7 transcripts, encoding zinc efflux transporters ZnT5, ZnT6, ZnT7, localized on endoplasmic reticulum membranes, might be part of a coordinated transcriptional program associated to the increased activity of the early secretory pathway, while transcriptional upregulation of several specific ZIP transporters (SLC39A6, SLC39A7, SLC39A9, SLC39A10, and SLC39A11) could contribute in meeting the increased demand of zinc in cancer cells. Moreover, exon-level analysis of SLC30A9, a nuclear receptor coactivator involved in the transcriptional regulation of Wnt-responsive genes, revealed the differential expression of alternative transcripts in CRC and normal colonic mucosa.


Assuntos
Proteínas de Transporte de Cátions/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Idoso , Neoplasias da Mama/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição , Via de Sinalização Wnt/genética , Zinco/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...