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1.
Int J Mol Sci ; 20(17)2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31454948

RESUMO

The transmembrane (TM) proteins are gateways for molecular transport across the cell membrane that are often selected as potential targets for drug design. The bilitranslocase (BTL) protein facilitates the uptake of various anions, such as bilirubin, from the blood into the liver cells. As previously established, there are four hydrophobic transmembrane segments (TM1-TM4), which constitute the structure of the transmembrane channel of the BTL protein. In our previous studies, the 3D high-resolution structure of the TM2 and TM3 transmembrane fragments of the BTL in sodium dodecyl sulfate (SDS) micellar media were solved using Nuclear Magnetic Resonance (NMR) spectroscopy and molecular dynamics simulations (MD). The high-resolution 3D structure of the fourth transmembrane region (TM4) of the BTL was evaluated using NMR spectroscopy in two different micellar media, anionic SDS and zwitterionic DPC (dodecylphosphocholine). The presented experimental data revealed the existence of an α -helical conformation in the central part of the TM4 in both micellar media. In the case of SDS surfactant, the α -helical conformation is observed for the Pro258-Asn269 region. The use of the zwitterionic DPC micelle leads to the formation of an amphipathic α -helix, which is characterized by the extension of the central α -helix in the TM4 fragment to Phe257-Thr271. The complex character of the dynamic processes in the TM4 peptide within both surfactants was analyzed based on the relaxation data acquired on 15 N and 31 P isotopes. Contrary to previously published and present observations in the SDS micelle, the zwitterionic DPC environment leads to intensive low-frequency molecular dynamic processes in the TM4 fragment.


Assuntos
Ceruloplasmina/química , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ceruloplasmina/metabolismo , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/metabolismo , Micelas , Peptídeos/química , Peptídeos/metabolismo , Relação Estrutura-Atividade
2.
Molecules ; 24(5)2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30818768

RESUMO

Phenols are the most abundant naturally accessible antioxidants present in a human normal diet. Since numerous beneficial applications of phenols as preventive agents in various diseases were revealed, the evaluation of phenols bioavailability is of high interest of researchers, consumers and drug manufacturers. The hydrophilic nature of phenols makes a cell membrane penetration difficult, which imply an alternative way of uptake via membrane transporters. However, the structural and functional data of membrane transporters are limited, thus the in silico modelling is really challenging and urgent tool in elucidation of transporter ligands. Focus of this research was a particular transporter bilitranslocase (BTL). BTL has a broad tissue expression (vascular endothelium, absorptive and excretory epithelia) and can transport wide variety of poly-aromatic compounds. With available BTL data (pKi [mmol/L] for 120 organic compounds) a robust and reliable QSAR models for BTL transport activity were developed and extrapolated on 300 phenolic compounds. For all compounds the transporter profiles were assessed and results show that dietary phenols and some drug candidates are likely to interact with BTL. Moreover, synopsis of predictions from BTL models and hits/predictions of 20 transporters from Metrabase and Chembench platforms were revealed. With such joint transporter analyses a new insights for elucidation of BTL functional role were acquired. Regarding limitation of models for virtual profiling of transporter interactions the computational approach reported in this study could be applied for further development of reliable in silico models for any transporter, if in vitro experimental data are available.


Assuntos
Membrana Celular/enzimologia , Ceruloplasmina/metabolismo , Simulação por Computador , Fenóis/metabolismo , Transporte Biológico , Transporte Biológico Ativo , Bases de Dados de Produtos Farmacêuticos , Humanos
3.
Biochim Biophys Acta ; 1828(11): 2609-19, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23774522

RESUMO

Membrane proteins represent about a third of the gene products in most organisms, as revealed by the genome sequencing projects. They account for up to two thirds of known drugable targets, which emphasizes their critical pharmaceutical importance. Here we present a study on bilitranslocase (BTL) (TCDB 2.A.65), a membrane protein primarily involved in the transport of bilirubin from blood to liver cells. Bilitranslocase has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its 3D structure. However, at present, only a limited knowledge is available beyond the primary structure of BTL. It has been recently confirmed experimentally that one of the four computationally predicted transmembrane segments of bilitranslocase, TM3, has a helical structure with hydrophilic amino acid residues oriented towards one side, which is typical for transmembrane domains of membrane proteins. In this study we confirmed by the use of multidimensional NMR spectroscopy that the second transmembrane segment, TM2, also appears in a form of α-helix. The stability of this polypeptide chain was verified by molecular dynamics (MD) simulation in dipalmitoyl phosphatidyl choline (DPPC) and in sodium dodecyl sulfate (SDS) micelles. The two α-helices, TM2 corroborated in this study, and TM3 confirmed in our previous investigation, provide reasonable building blocks of a potential transmembrane channel for transport of bilirubin and small hydrophilic molecules, including pharmaceutically active compounds.


Assuntos
Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Sequência de Aminoácidos , Transporte Biológico Ativo , Ceruloplasmina , Dicroísmo Circular , Micelas , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Conformação Proteica , Dodecilsulfato de Sódio
4.
Radiol Oncol ; 47(2): 128-37, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23801909

RESUMO

BACKGROUND: Bilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues. MATERIALS AND METHODS: Mice were immunized with a multi-antigen peptide corresponding to segment 65-75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase. RESULTS: On the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use. CONCLUSIONS: In this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures.

5.
J Nutr Biochem ; 51: 8-15, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29078076

RESUMO

Olive oil vascular benefits have been attributed to hydroxytyrosol (HT). However, HT biological actions are still debated because it is extensively metabolized into glucuronides (GCs). The aim of this study was to test HT and GC vasculoprotective effects and the underlying mechanisms using aorta rings from 8-week-old male Wistar rats. In the absence of oxidative stress, incubation with 100 µM HT or GC for 5 min did not exert any vasorelaxing effect and did not influence the vascular function. Conversely, in condition of oxidative stress [upon incubation with 500 µM tert-butylhydroperoxide (t-BHP) for 30 min], preincubation with HT or GC improved acetylcholine-induced vasorelaxation compared with untreated samples (no t-BHP). This protective effect was lost for GC, but not for HT, when a washing step (15 min) was introduced between preincubation with HT or GC and t-BHP addition, suggesting that only HT enters the cells. In agreement, bilitranslocase inhibition with 100 µM phenylmethanesulfonyl fluoride for 20 min reduced significantly HT, but not GC, effect on the vascular function upon stress induction. Moreover, GC protective effect (improvement of endothelium-dependent relaxation in response to acetylcholine) in oxidative stress conditions was reduced by preincubation of aorta rings with 300 µM D-saccharolactone to inhibit ß-glucuronidase, which can deconjugate polyphenols. Finally, only HT was detected by high-pressure liquid chromatography in aorta rings incubated with GC and t-BHP. These results suggest that, in conditions of oxidative stress, GC can be deconjugated into HT that is transported through the cell membrane by bilitranslocase to protect vascular function.


Assuntos
Antioxidantes/metabolismo , Ceruloplasmina/metabolismo , Endotélio Vascular/metabolismo , Glucuronidase/metabolismo , Glucuronídeos/metabolismo , Estresse Oxidativo , Álcool Feniletílico/análogos & derivados , Animais , Antioxidantes/química , Aorta Torácica , Transporte Biológico Ativo/efeitos dos fármacos , Ceruloplasmina/antagonistas & inibidores , Suplementos Nutricionais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Inibidores Enzimáticos/farmacologia , Ácido Glucárico/análogos & derivados , Ácido Glucárico/farmacologia , Glucuronidase/antagonistas & inibidores , Glucuronídeos/química , Técnicas In Vitro , Masculino , Moduladores de Transporte de Membrana/farmacologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Álcool Feniletílico/química , Álcool Feniletílico/metabolismo , Fluoreto de Fenilmetilsulfonil/farmacologia , Ratos Wistar , Doenças Vasculares/enzimologia , Doenças Vasculares/metabolismo , Doenças Vasculares/prevenção & controle , Vasodilatação/efeitos dos fármacos , terc-Butil Hidroperóxido/farmacologia
6.
Comput Struct Biotechnol J ; 15: 232-242, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28228927

RESUMO

The structural and functional details of transmembrane proteins are vastly underexplored, mostly due to experimental difficulties regarding their solubility and stability. Currently, the majority of transmembrane protein structures are still unknown and this present a huge experimental and computational challenge. Nowadays, thanks to X-ray crystallography or NMR spectroscopy over 3000 structures of membrane proteins have been solved, among them only a few hundred unique ones. Due to the vast biological and pharmaceutical interest in the elucidation of the structure and the functional mechanisms of transmembrane proteins, several computational methods have been developed to overcome the experimental gap. If combined with experimental data the computational information enables rapid, low cost and successful predictions of the molecular structure of unsolved proteins. The reliability of the predictions depends on the availability and accuracy of experimental data associated with structural information. In this review, the following methods are proposed for in silico structure elucidation: sequence-dependent predictions of transmembrane regions, predictions of transmembrane helix-helix interactions, helix arrangements in membrane models, and testing their stability with molecular dynamics simulations. We also demonstrate the usage of the computational methods listed above by proposing a model for the molecular structure of the transmembrane protein bilitranslocase. Bilitranslocase is bilirubin membrane transporter, which shares similar tissue distribution and functional properties with some of the members of the Organic Anion Transporter family and is the only member classified in the Bilirubin Transporter Family. Regarding its unique properties, bilitranslocase is a potentially interesting drug target.

7.
Toxicol Lett ; 225(2): 305-10, 2014 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-24374050

RESUMO

Bilitranslocase (BTL) is a plasma membrane carrier that transports organic anions of physiological and pharmacological interest. It is expressed in basolateral plasma membrane of kidney and liver. BTL has been recently described as a marker of transition from normal tissue to its neoplastic transformation in human kidney. Inorganic mercury is a major environmental contaminant that produces many toxic effects. Previous reports have described an interaction between BTL and mercuric ions. This study was designed to evaluate the renal and hepatic expression of BTL in rats exposed to a nephrotoxic and hepatotoxic dose of HgCl2. Male rats were treated with a single injection of HgCl2 at a dose of 4mg/kg body wt, i.p. (HgCl2 group). Control rats received the vehicle alone (Control group). Studies were carried out 18h after injection. Afterwards, the kidneys and livers were excised and processed for histopathological studies or immunoblot (homogenates and crude membranes) techniques. In rats treated with HgCl2, immunoblotting showed a significant decrease in the abundance of BTL in homogenates and plasma membranes from kidney and liver. BTL decrease of expression might reflect the grade of damage in renal tubule cells and in hepatocytes. Thus, BTL might be postulated as a new biomarker of tissue toxicity induced by mercury.


Assuntos
Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Cloreto de Mercúrio/toxicidade , Animais , Aspartato Aminotransferases/sangue , Biomarcadores/metabolismo , Peso Corporal/efeitos dos fármacos , Ceruloplasmina , Regulação da Expressão Gênica , Rim/enzimologia , Fígado/enzimologia , Masculino , Proteínas de Membrana/genética , Mercúrio/urina , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Wistar
8.
SAR QSAR Environ Res ; 25(11): 853-72, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25337672

RESUMO

Membrane transport proteins are essential for cellular uptake of numerous salts, nutrients and drugs. Bilitranslocase is a transporter, specific for water-soluble organic anions, and is the only known carrier of nucleotides and nucleotide-like compounds. Experimental data of bilitranslocase ligand specificity for 120 compounds were used to construct classification models using counter-propagation artificial neural networks (CP-ANNs) and support vector machines (SVMs). A subset of active compounds with experimentally determined transport rates was used to build predictive QSAR models for estimation of transport rates of unknown compounds. Several modelling methods and techniques were applied, i.e. CP-ANN, genetic algorithm, self-organizing mapping and multiple linear regression method. The best predictions were achieved using CP-ANN coupled with a genetic algorithm, with the external validation parameter QV(2) of 0.96. The applicability domains of the models were defined to determine the chemical space in which reliable predictions can be obtained. The models were applied for the estimation of bilitranslocase transport activity for two sets of pharmaceutically interesting compounds, antioxidants and antiprions. We found that the relative planarity and a high potential for hydrogen bond formation are the common structural features of anticipated substrates of bilitranslocase. These features may serve as guidelines in the design of new pharmaceuticals transported by bilitranslocase.


Assuntos
Antioxidantes/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Príons/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Transporte Biológico Ativo , Ceruloplasmina , Modelos Lineares , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Chaperonas Moleculares/química , Redes Neurais de Computação , Preparações Farmacêuticas/metabolismo , Máquina de Vetores de Suporte
9.
World J Gastroenterol ; 18(44): 6387-97, 2012 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23197884

RESUMO

Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct. The absorption, distribution and elimination of drugs are impaired during this pathology. Prolonged cholestasis may alter both liver and kidney function. Lactam antibiotics, diuretics, non-steroidal anti-inflammatory drugs, several antiviral drugs as well as endogenous compounds are classified as organic anions. The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds. It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions. The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis, such as multidrug resistance-associated protein 2, organic anion transporting polypeptide 1, organic anion transporter 3, bilitranslocase, bromosulfophthalein/bilirubin binding protein, organic anion transporter 1 and sodium dependent bile salt transporter. The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.


Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase Extra-Hepática/metabolismo , Icterícia Obstrutiva/metabolismo , Rim/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Adaptação Fisiológica , Animais , Transporte Biológico , Humanos , Preparações Farmacêuticas/metabolismo , Farmacocinética
10.
Plant Signal Behav ; 3(9): 626-32, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19513253

RESUMO

Flavonoids are a group of secondary metabolites widely distributed in plants that represent a huge portion of the soluble phenolics present in grapevine (Vitis vinifera L.). These compounds play different physiological roles and are often involved in protection against biotic and abiotic stress. Even if the flavonoid biosynthetic pathways have been largely characterized, the mechanisms of their transport and accumulation in cell wall and vacuole are still not completely understood. This review analyses the known mechanisms of flavonoid uptake and accumulation in grapevine, with reference to the transport models and membrane carrier proteins described in other plant species. The effect of different environmental factors on flavonoid biosynthesis and transporters is also discussed.

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