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1.
Talanta ; 206: 120177, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514882

RESUMO

Two highly selective OFF-ON isomer fluorescent probes (1 and 2) for homo-/cysteine were designed and synthesized. The pyrene modified tetraphenylethylene derivative with AIE was used as luminescent group while maleimide was used as recognition group. These two isomer probes were found to be nearly nonfluorescent when treated with GSH. However, upon interaction with Cys or Hcy, the fluorescence was enhanced by 2000 folds in a wide pH range from 3 to 10. Experimental results and DFT calculation have demonstrated that the fluorescence OFF-ON switch of such thiol probes is resulted from the termination of the PET (photo-induced electron transfer) effect through the Michael addition reaction of maleimide unit and thiols. In addition, probe 1 and 2 exhibit excellent selectivity and sensitivity towards Cys, Hcy over GSH and other amino acids, which was confirmed by mass MS. We suggested that Michael addition reaction of these probes with GSH was prevented because of the stereo-hindrance effect. Furthermore, these two isomer probes were successfully used for imaging biothiols in living H1299 lung cancer cells.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Glutationa/química , Homocisteína/análise , Linhagem Celular Tumoral , Cisteína/química , Teoria da Densidade Funcional , Fluorescência , Corantes Fluorescentes/síntese química , Homocisteína/química , Humanos , Maleimidas/síntese química , Maleimidas/química , Microscopia de Fluorescência/métodos , Modelos Químicos , Estilbenos/síntese química , Estilbenos/química
2.
Analyst ; 144(17): 5075-5080, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31305811

RESUMO

Herein, we report the design of a novel fluorescent probe consisting of a naphthalimide fluorophore and a silicone small molecule for the reversible detection of hypochlorous acid and biothiol amino acids. The response mechanism of BSi-1 is based on the concept of the S-based oxidation/reduction. The probe was found to be suitable for imaging HOCl in HeLa, RAW 264.7 cells and zebrafish, demonstrating its utility in biological applications.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Homocisteína/análise , Ácido Hipocloroso/análise , Naftalimidas/química , Animais , Cisteína/química , Glutationa/química , Células HeLa , Homocisteína/química , Humanos , Limite de Detecção , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Estrutura Molecular , Oxirredução , Células RAW 264.7 , Peixe-Zebra
3.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 592-604, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205021

RESUMO

The equilibrium between phosphorylation and dephosphorylation is one of the most important processes that takes place in living cells. Human phosphoserine phosphatase (hPSP) is a key enzyme in the production of serine by the dephosphorylation of phospho-L-serine. It is directly involved in the biosynthesis of other important metabolites such as glycine and D-serine (a neuromodulator). hPSP is involved in the survival mechanism of cancer cells and has recently been found to be an essential biomarker. Here, three new high-resolution crystal structures of hPSP (1.5-2.0 Å) in complexes with phosphoserine and with serine, which are the substrate and the product of the reaction, respectively, and in complex with a noncleavable substrate analogue (homocysteic acid) are presented. New types of interactions take place between the enzyme and its ligands. Moreover, the loop involved in the open/closed state of the enzyme is fully refined in a totally unfolded conformation. This loop is further studied through molecular-dynamics simulations. Finally, all of these analyses allow a more complete reaction mechanism for this enzyme to be proposed which is consistent with previous publications on the subject.


Assuntos
Homocisteína/análogos & derivados , Monoéster Fosfórico Hidrolases/química , Fosfosserina/química , Serina/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X/métodos , Escherichia coli , Homocisteína/química , Humanos , Ligantes , Simulação de Dinâmica Molecular , Fosfosserina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Serina/metabolismo
4.
Electrophoresis ; 40(15): 1913-1920, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30892703

RESUMO

The enantiomeric separation of 9-fluorenylmethoxycarbonyl chloride (FMOC)-homocysteine (Hcy) by CE was investigated using γ-CD and the chiral ionic liquid (R)-(1-hydroxybutan-2-yl)(trimethyl)azanium-bis(trifluoromethanesulfon)imidate (also called (R)-N,N,N-trimethyl-2-aminobutanol-bis(trifluoromethane-sulfon)imidate) (EtCholNTf2 ) as chiral selectors. Using 2 mM γ-CD and 5 mM EtCholNTf2 in 50 mM borate buffer (pH 9), FMOC-Hcy enantiomers were separated with a resolution value of 3.8. A reversal in the enantiomer migration order in comparison with the single use of γ-CD in the separation buffer was obtained. Then, NMR experiments were carried out to elucidate the interactions taking place in the enantiomeric separation of FMOC-Hcy. NMR analyses highlighted the formation of an inclusion complex since the hydrophobic group of FMOC-Hcy was inserted into the γ-CD cavity. Moreover, interactions between EtCholNTf2 and γ-CD were also observed, suggesting that the chiral ionic liquid would also enter the cavity of the γ-CD.


Assuntos
Eletroforese Capilar/métodos , Homocisteína/isolamento & purificação , Líquidos Iônicos/química , Espectroscopia de Ressonância Magnética/métodos , gama-Ciclodextrinas/química , Homocisteína/análise , Homocisteína/química , Imidazóis/química , Estereoisomerismo
5.
Biochim Biophys Acta Gen Subj ; 1863(5): 941-949, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30853337

RESUMO

BACKGROUND: Elevated homocysteine is epidemiologically related to insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling. However, the effect of homocysteine on PTP1B remains unclear. METHODS: S-homocysteinylated PTP1B was identified by LC-ESI-MS/MS. The ability of thioredoxin system to recover active PTP1B from S-homocysteinylated PTP1B was confirmed by RNA interference. To address the mechanism for homocysteine to affect PTP1B activity, we performed 5-IAF insertion, activity assays, Western blotting, co-immunoprecipitation and glucose uptake experiments. RESULTS: The thiol-containing form of homocysteine (HcySH) suppressed phosphorylation of insulin receptor-ß subunit, but enhanced PTP1B activity. This phenomenon was partially related to the fact that HcySH promoted PTP1B expression. Although the disulfide-bonded form of homocysteine (HSSH) modified PTP1B to form an inactive S-homocysteinylated PTP1B, HcySH-induced increase in the activities of cellular thioredoxin and thioredoxin reductase, components of thioredoxin system, could recover active PTP1B from S-homocysteinylated PTP1B. Thioredoxin system transferred electrons from NADPH to S-homocysteinylated PTP1B, regenerating active PTP1B in vitro and in hepatocytes. The actions of HcySH were also related with decrease in hepatic glucose uptake. CONCLUSIONS: The effect of HcySH/HSSH on PTP1B activity depends, at least partially, on the ratio of active PTP1B and S-homocysteinylated PTP1B. High HcySH-induced an increase in thioredoxin system activity is beneficial to de-S-homocysteinylation and is good for PTP1B activity. GENERAL SIGNIFICANCE: Our data provide a novel insight into post-translational regulation of PTP1B, and expand the biological functions of thioredoxin system.


Assuntos
Hepatócitos/química , Homocisteína/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Homocisteína/análogos & derivados , Homocisteína/química , Humanos , Proteína Tirosina Fosfatase não Receptora Tipo 1/química
6.
Chem Commun (Camb) ; 55(25): 3654-3657, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30855056

RESUMO

Herein, we present a serendipitously discovered chemoselective labelling of protein N-homocysteinylation with bioorthogonal azide probes. The reaction proceeds rapidly under alkaline and heating conditions. Our experiments suggest that azides can be converted to aldehydes in situ catalyzed by heme(ii), followed by a condensation with protein N-homocysteinylation to afford stable 1,3-thiazines.


Assuntos
Azidas/química , Heme/química , Homocisteína/análogos & derivados , Proteínas/química , Animais , Biotina/química , Catálise , Bovinos , Homocisteína/química , Concentração de Íons de Hidrogênio , Mioglobina/química , Mioglobina/metabolismo , Peptídeos/análise , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Espectrometria de Massas em Tandem , Temperatura
7.
Talanta ; 195: 197-203, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625532

RESUMO

Biological thiols (biothiols), such as glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), play a vital role in the process of reversible redox reactions in physiological systems. In this work, flow cytometry-based fluorescent sensor is for the first time developed for the detection of biothiols in a fluorescence "turn on" manner. The probe which we name "Polystyrene/Quantum Dots/Gold Nanoparticles" or (PS/QDs/Au) is constructed by immobilizing QDs onto the surface of PS microbeads to obtain fluorescent microbeads. The probe (PS/QDs/Au) is constructed by immobilizing QDs onto the surface of PS microbeads to obtain fluorescent microbeads, followed by gold NPs absorption through electrostatic interaction to quench their fluorescence. In the presence of biothiols, the fluorescence of our probe can be restored in less than 5 min, and the detection limits for GSH, Cys and Hcy are 0.5 µM, 0.1 µM and 0.3 µM, respectively. Most importantly, the fluorescence signal of each of our probe microbeads can be collected individually by flow cytometry, realizing single microbead-based biothiols detection for the first time. Moreover, the probe is successfully applied to imaging of intracellular biothiols in A549 cells, demonstrating its potential in biological application.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Homocisteína/análise , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Cisteína/química , Citometria de Fluxo , Fluorescência , Corantes Fluorescentes/toxicidade , Glutationa/química , Ouro/química , Ouro/toxicidade , Homocisteína/química , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Microesferas , Poliestirenos/química , Poliestirenos/toxicidade , Pontos Quânticos/química , Pontos Quânticos/toxicidade
8.
Talanta ; 195: 281-289, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625544

RESUMO

Biothiols, such as glutathione (GSH), homocysteine (Hcy) and cysteine (Cys), are important biomarkers and play crucial roles in many physiological processes. Thus, the detection of biothiols is highly important for early diagnosis of diseases and evaluation of disease progression. Herein, new types of BODIPY-based fluorescent probes (probe 1, probe 2 and probe 3) capable of cysteine (Cys)/homocysteine (Hcy) sensing with high selectivity over other amino acids were developed. In addition, we further studied the influence of different electronegativity substituents on these probes to sensing Cys/Hcy. Ultimately, we concluded that the electron withdrawing group on probe 1 can accelerate the probe response to Cys/Hcy, and probe 1 was successfully applied for selective imaging Cys/Hcy in living cells.


Assuntos
Compostos de Boro/química , Cisteína/análise , Corantes Fluorescentes/química , Homocisteína/análise , Cisteína/química , Células HeLa , Homocisteína/química , Humanos , Espectrometria de Fluorescência
9.
Talanta ; 196: 145-152, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30683344

RESUMO

A long-wavelength fluorescent probe NR-CY was developed for simultaneous identification of cysteine/glutathione and sulphide by combining the derivative of Nile red with 7-nitrobenzofurazan. The response of NR-CY to thiols is regulated by intramolecular charge transfer and photoinduced electron transfer mechanisms. For sulphide at 560 nm, cysteine at 475 nm and glutathione at 425 nm, different absorbance increases can be observed. NR-CY can detect cysteine at fluorescence emission 543 nm and distinguish sulphide from other analytes by kinetic experiments at 636 nm. The probe showed a rapid response to these thiols (cysteine was 90 s and sulphide was 30 s). In addition, NR-CY has been successfully applied to live MCF-7 cell imaging.


Assuntos
4-Cloro-7-nitrobenzofurazano/química , Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Homocisteína/análise , Oxazinas/química , Cisteína/química , Glutationa/química , Homocisteína/química , Humanos , Células MCF-7
10.
Talanta ; 194: 717-722, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30609596

RESUMO

A new simple and facile fluorescent ratiometric probe (probe 1) has been designed for the detection of cysteine (Cys). Probe 1 as the fluorophore contains a typical excited-state intramolecular proton transfer (ESIPT) dye. In probe 1 to restore the ESIPT process, the group of acrylate which acts as the recognition unit can block the ESIPT process and can be selectively achieved by Cys, which makes the probe as the ratiometric fluorescent detection for Cys in aqueous solution. This probe shows highly selectivity towards Cys over other biothiols including glutathione (GSH) and homocysteine (Hcy) because of specific cyclization between Cys and acrylate group, and having a detection limit of 42.3 nM. In addition, the experiment of cell imaging shows that probe 1 possesses low cytotoxicity and excellent cell permeability towards the living cells, and has been successfully applied to the ratiometric imaging not only for the endogenous but also for the exogenous cysteine in the living cells effectively.


Assuntos
Cisteína/análise , Cisteína/química , Corantes Fluorescentes/química , Limite de Detecção , Prótons , Sobrevivência Celular , Ciclização , Corantes Fluorescentes/toxicidade , Glutationa/química , Células HeLa , Homocisteína/química , Humanos , Concentração de Íons de Hidrogênio , Imagem Óptica , Fatores de Tempo , Água/química
11.
J Microbiol Biotechnol ; 29(1): 114-126, 2019 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-30518019

RESUMO

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH2CH2SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.


Assuntos
Bioensaio/métodos , Hidrocarbonetos Aromáticos com Pontes/química , Homocisteína/análise , Homocisteína/química , Imidazóis/química , Anticorpos Monoclonais/imunologia , Cistationina gama-Liase/química , Cisteína/química , Ácido Ditionitrobenzoico/química , Epitopos/imunologia , Homocisteína/imunologia , Humanos , Modelos Moleculares , Estrutura Molecular , Reagentes de Sulfidrila/química
12.
Physiol Rev ; 99(1): 555-604, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30427275

RESUMO

Epidemiological studies established that elevated homocysteine, an important intermediate in folate, vitamin B12, and one carbon metabolism, is associated with poor health, including heart and brain diseases. Earlier studies show that patients with severe hyperhomocysteinemia, first identified in the 1960s, exhibit neurological and cardiovascular abnormalities and premature death due to vascular complications. Although homocysteine is considered to be a nonprotein amino acid, studies over the past 2 decades have led to discoveries of protein-related homocysteine metabolism and mechanisms by which homocysteine can become a component of proteins. Homocysteine-containing proteins lose their biological function and acquire cytotoxic, proinflammatory, proatherothrombotic, and proneuropathic properties, which can account for the various disease phenotypes associated with hyperhomocysteinemia. This review describes mechanisms by which hyperhomocysteinemia affects cellular proteostasis, provides a comprehensive account of the biological chemistry of homocysteine-containing proteins, and discusses pathophysiological consequences and clinical implications of their formation.


Assuntos
Doenças Cardiovasculares/metabolismo , Homocisteína/metabolismo , Hiper-Homocisteinemia/metabolismo , Vitamina B 12/metabolismo , Animais , Ácido Fólico/metabolismo , Homocisteína/química , Humanos , Fatores de Risco
13.
J Pharm Biomed Anal ; 164: 442-451, 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30447532

RESUMO

An ultra-high performance hydrophilic interaction liquid chromatography - triple quadrupole tandem mass spectrometry method was developed for the determination of biologically important thiols, namely cysteine, homocysteine, cysteinyl-glycine, glutathione, in rat plasma. The sample preparation procedure as well as the analytical method were comprehensively optimized and subsequently validated. An optimum sample preparation protocol was based on the simple and fast derivatization of the thiols with new derivatization reagent, N-phenylmaleimide, enabling highly selective and sensitive quantification in plasma matrices. The method, characterized by favourable performance parameters and meeting the FDA criteria for biomedical analysis, was successfully applied for monitoring the concentration levels of the selected thiols in the samples from transgenic rat model for tauopathy. The study revealed significant changes in homocysteine and glutathione levels related to tauopathy while other thiols did not indicate such relationship. Indeed, these findings could play an important role in further understanding of tauopathy process in the brain. Moreover, the proposed highly effective, reliable and robust analytical protocol can be easily adapted for other thiol compounds, spreading its application range in this biomedical field.


Assuntos
Fracionamento Químico/métodos , Glutationa/sangue , Homocisteína/sangue , Tauopatias/sangue , Animais , Fracionamento Químico/instrumentação , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Cisteína/sangue , Cisteína/química , Dipeptídeos/sangue , Dipeptídeos/química , Modelos Animais de Doenças , Glutationa/química , Homocisteína/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Maleimidas/química , Ratos , Ratos Endogâmicos SHR , Ratos Transgênicos , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos , Tauopatias/diagnóstico , Tauopatias/genética , Tauopatias/patologia , Proteínas tau/genética
14.
Cell Rep ; 25(2): 398-412.e6, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30304680

RESUMO

Colorectal cancer (CRC) onset is profoundly affected by Western diet. Here, we report that high-fat (HF) diet-induced, organ-specific colonic lysine homocysteinylation (K-Hcy) increase might promote CRC onset by impeding DNA damage repair. HF chow induced elevated methionyl-tRNA synthetase (MARS) expression and K-Hcy levels and DNA damage accumulation in the mouse and rat colon, resulting in a phenotype identical to that of CRC tissues. Moreover, the increased copy number of MARS, whose protein product promotes K-Hcy, correlated with increased CRC risk in humans. Mechanistically, MARS preferentially bound to and modified ataxia-telangiectasia and Rad3-related protein (ATR), inhibited ATR and its downstream effectors checkpoint kinase-1 and p53, and relieved cell-cycle arrest and decreased DNA damage-induced apoptosis by disrupting the binding of ATR-interacting protein to ATR. Inhibiting K-Hcy by targeting MARS reversed these effects and suppressed oncogenic CRC cell growth. Our study reveals a mechanism of Western-diet-associated CRC and highlights an intervention approach for reversing diet-induced oncogenic effects.


Assuntos
Neoplasias do Colo/patologia , Dano ao DNA , Reparo do DNA , Dieta Hiperlipídica/efeitos adversos , Homocisteína/química , Lisina/química , Neoplasias Retais/patologia , Animais , Apoptose , Estudos de Casos e Controles , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Processamento de Proteína Pós-Traducional , Ratos , Ratos Wistar , Neoplasias Retais/genética , Neoplasias Retais/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Anal Chem ; 90(21): 12944-12950, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30280562

RESUMO

The expansion of electrochemical sensors to biomedical applications at point of care requires these sensors to undergo analysis without any pretreatment or extraction. This poses a major challenge for all electrochemical sensors including electrochemiluminescent (ECL)-based sensors. ECL offers many advantages for biomedical applications; however, obtaining results from complex matrixes has proven to be a large hurdle for the application of ECL sensors within this field. This work demonstrates the potential of cathodic ECL to detect and quantify homocysteine (Hcy) with a 0.1 nM limit of detection, which is associated with hyperhomocysteinemia, in blood. This near-infrared quantum dot (NIR QD)-based ECL sensor displays good linearity allowing for rapid detection and providing a basis for exploitation of ECL-based sensors for biomedical diagnostics utilizing Hcy as a model cathodic coreactant. This work will lay the foundations for future developments in biosensing and imaging fields and stands as an initial proof of concept for the utilization of cathodic ECL technologies for biomedical applications once the limits of detection within clinically relevant levels has been achieved. This work illustrates the potential of cathodic ECL sensors, using Hcy as a model complex, for the detection of biomolecules.


Assuntos
Biomarcadores/sangue , Técnicas Eletroquímicas/métodos , Homocisteína/sangue , Medições Luminescentes/métodos , Pontos Quânticos/química , Animais , Biomarcadores/química , Compostos de Cádmio/química , Bovinos , Técnicas Eletroquímicas/instrumentação , Eletrodos , Homocisteína/química , Luminescência , Oxirredução , Compostos de Selênio/química , Sulfetos/química , Telúrio/química , Compostos de Zinco/química
16.
FEBS Lett ; 592(20): 3399-3413, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30194685

RESUMO

Glutamate racemases (GR) catalyze the racemization of d- and l-glutamate and are targets for the development of antibiotics. We demonstrate that GR from the periodontal pathogen Fusobacterium nucleatum (FnGR) catalyzes the racemization of d-homocysteic acid (d-HCA), while l-HCA is a poor substrate. This enantioselectivity arises because l-HCA perturbs FnGR's monomer-dimer equilibrium toward inactive monomer. The inhibitory effect of l-HCA may be overcome by increasing the total FnGR concentration or by adding glutamate, but not by blocking access to the active site through site-directed mutagenesis, suggesting that l-HCA binds at an allosteric site. This phenomenon is also exhibited by GR from Bacillus subtilis, suggesting that enantiospecific, "substrate"-induced dissociation of oligomers to form inactive monomers may furnish a new inhibition strategy.


Assuntos
Isomerases de Aminoácido/química , Proteínas de Bactérias/química , Homocisteína/análogos & derivados , Estrutura Quaternária de Proteína , Sítio Alostérico , Isomerases de Aminoácido/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Fusobacterium nucleatum/enzimologia , Homocisteína/química , Homocisteína/metabolismo , Cinética , Estereoisomerismo , Especificidade por Substrato
17.
FEBS J ; 285(20): 3801-3814, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30103295

RESUMO

Many patients under therapy with recombinant human erythropoietin (rhuEPO) show resistance to the treatment, an effect likely associated with the accumulation of tissue factors, especially in renal and cardiovascular diseases. Hyperhomocysteinemia due to high serum levels of homocysteine has been suggested among the risk factors in those pathologies. Its main effect is the N-homocysteinylation of proteins due to the interaction between the highly reactive homocysteine thiolactone (HTL) and lysine residues. The aim of this study was to evaluate the effect of N-homocysteinylation on the erythropoietic and antiapoptotic abilities of EPO, which can be a consequence of structural changes in the modified protein. We found that both cellular functions were altered in the presence of HTL-EPO. A decreased net positive charge of HTL-EPO was detected by capillary zone electrophoresis, while analysis of polyacrylamide gel electropherograms suggested formation of aggregates. Far-UV spectra, obtained by Circular Dichroism Spectroscopy, indicated a switch of the protein's secondary structure from α-helix to ß-sheet structures. Results of Congo red and Thioflavin T assays confirm the formation of repetitive ß-sheet structures, which may account for aggregates. Accordingly, Dynamic Light Scattering analysis showed a markedly larger radius of the HTL-EPO structures, supporting the formation of soluble oligomers. These structural changes might interfere with the conformational adaptations necessary for efficient ligand-receptor interaction, thus affecting the proliferative and antiapoptotic functions of EPO. The present findings may contribute to explain the resistance exhibited by patients with cardio-renal syndrome to treatment with rhuEPO, as a consequence of structural modifications due to protein N-homocysteinylation.


Assuntos
Apoptose , Proliferação de Células , Eritropoetina/química , Homocisteína/análogos & derivados , Lisina/química , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Células Cultivadas , Eritropoetina/metabolismo , Homocisteína/química , Humanos , Megacariócitos/metabolismo , Megacariócitos/patologia , Ligação Proteica
18.
J Chromatogr A ; 1568: 222-228, 2018 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-30025612

RESUMO

The enantioseparation of the non-protein amino acid homocysteine by CE was investigated in this article using eleven neutral CDs and five chiral ionic liquids as chiral selectors. Using a previous derivatization step with FMOC and the subsequent separation under neutral conditions, homocysteine enantiomers were only separated when γ-CD or (R)-N,N,N-trimethyl-2-aminobutanol-bis(trifluoromethane-sulfon)imidate (EtCholNTf2) were employed as sole chiral selectors in the separation buffer. On the one hand, γ-CD gave rise to the enantiomeric separation in 10min with a resolution value of 1.9, whereas EtCholNTf2 let to obtain a resolution value of 1.4 in more than 50min. Then, the evaluation of the combined use of both selectors was also carried out, resulting in a considerable increase in the Rs. The best enantioseparation for homocysteine was obtained when 10mM EtCholNTf2 was added to 50mM phosphate buffer (pH 7.0) containing 2mM γ-CD. In an attempt to discriminate specific chiral cation effect from the salt effect, the influence of adding LiNTf2 to the separation medium was also evaluated, resulting in lower resolution values for homocysteine when compared to those achieved with the addition of EtCholNTf2, indicating a synergistic effect between EtCholNTf2 and γ-CD. Interestingly, the enantiomer migration order changed depending on the use of a single chiral selector or dual systems. When EtCholNTf2 or γ-CD were employed as sole chiral selectors, D-enantiomer was the first-migrating enantiomer. However, an inversion in the migration order was observed when both selectors were combined in a dual system being the L-enantiomer the first-migrating one.


Assuntos
Eletroforese Capilar/métodos , Homocisteína/química , Homocisteína/isolamento & purificação , Líquidos Iônicos/química , gama-Ciclodextrinas/química , Tampões (Química) , Estereoisomerismo , Fatores de Tempo
19.
J Mol Model ; 24(7): 177, 2018 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-29943287

RESUMO

Substance P is a neurotransmitter or modulator in both the central and peripheral nervous systems. In this work, modifications of the lysine in SP by homocysteine and an acetyl group as well as the conformational dynamics of the native and modified SP peptides and their complexes with the NK1 receptor were studied via MD simulation. It was found that modifying SP stabilizes the peptide structure, but the modified SP peptides are less likely to bind to the NK1 receptor, so the resulting complexes are less stable. The RMSD of native SP (~0.33 nm) is about twice as large as that of the modified SP peptides (~0.18 nm), while the RMSD for the receptor complexed with native SP is ~0.3 nm, and that for the receptor complexed with either of the modified peptides is ~0.35 nm, which demonstrates the high stability of the modified SP peptides as well as the receptor complexed with native SP. Such behavior was also observed in other structural analyses. The binding free energies of the native and modified SP peptides with the NK1 receptor were also compared. The ΔGbind values for the binding of homocysteinylated SP to the NK1 receptor and the binding of the acetylated SP and native SP to the NK1 receptor were -38.89, -64.46, and - 264.52 kJ mol-1, respectively. Modification of the lysine of SP decreases the binding affinity of the peptide to the NK1 receptor. In other words, homocysteinylation or acetylation of SP leads to weaker interactions of the peptide with the NK1 receptor compared to those between native SP and NK1. We propose that this phenomenon leads to increased levels of homocysteinylated SP in plasma in many diseases such as breast cancer. Graphical abstract Substance P (SP) is a neuropeptide which binds to the NK1 receptor. SP is of great pharmacological interest, as agonists and antagonists of SP can potentially be used to treat many chronic diseases. Therefore, in this work, the lysine (LYS) in SP was theoretically modified with a homocysteine or acetyl group to explore the effects of such a modification on the binding affinity of this peptide with the NK1 receptor and the structural dynamics of the resulting complex.


Assuntos
Homocisteína/química , Simulação de Dinâmica Molecular , Receptores da Neurocinina-1/química , Substância P/química , Aminoácidos/química , Sítios de Ligação , Homocisteína/metabolismo , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Receptores da Neurocinina-1/metabolismo , Relação Estrutura-Atividade , Substância P/metabolismo
20.
Talanta ; 186: 110-118, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-29784337

RESUMO

Development of thiol-specific fluorescent probes with selectivity in different thiol compounds is more practical and significant than those without that capacity. In this work, a new quinoline-derived fluorophore, hydroxyl-substituted quinoline-benzo[d]oxazole 6 with high fluorescence quantum yield is synthesized and esterified with acrylic acid to afford two fluorescent probes, BQA-1 and BQA-2 for selectively discriminating Cys from Hcys/GSH based on conjugate addition-cyclization mechanism. BQA-1 exhibits a large ratiometric fluorescence response toward Cys in aqueous pH 7.4 solution with big emission peak-shifting from 383 nm to 518 nm, over 130 nm. The detection limit is determined to be as low as 0.59 µM. In contrast to BQA-1, BQA-2 whose acrylic ester moiety is further modified with pyridine group, displays a turn-on fluorescence response to Cys with detection limit of 0.98 µM. Both BQA-1 and BQA-2 have relatively weak response to another two biothiols, Hcys and GSH and nearly no response to other nucleophiles. Furthermore, the potential application for the detection of biothiols in living cells has been demonstrated by cell imaging experiment.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Glutationa/química , Homocisteína/química , Imagem Óptica , Quinolinas/química , Corantes Fluorescentes/síntese química , Humanos , Cinética , Estrutura Molecular , Quinolinas/síntese química , Células Tumorais Cultivadas
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