A Regulator Role for the ATP-Binding Cassette Subfamily C Member 6 Transporter in HepG2 Cells: Effect on the Dynamics of Cell-Cell and Cell-Matrix Interactions.

There is growing evidence that various ATP-binding cassette (ABC) transporters contribute to the growth and development of tumors, but relatively little is known about how the ABC transporter family behaves in hepatocellular carcinoma (HCC), one of the most common cancers worldwide. Cellular model studies have shown that ABCC6, which belongs to the ABC subfamily C (ABCC), plays a role in the cytoskeleton rearrangement and migration of HepG2 hepatocarcinoma cells, thus highlighting its role in cancer biology. Deep knowledge on the molecular mechanisms underlying the observed results could provide therapeutic insights into the tumors in which ABCC6 is modulated. In this study, differential expression levels of mRNA transcripts between ABCC6-silenced HepG2 and control groups were measured, and subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Real-Time PCR and Western blot analyses confirmed bioinformatics; functional studies support the molecular mechanisms underlying the observed effects. The results provide valuable information on the dysregulation of fundamental cellular processes, such as the focal adhesion pathway, which allowed us to obtain detailed information on the active role that the down-regulation of ABCC6 could play in the biology of liver tumors, as it is involved not only in cell migration but also in cell adhesion and invasion.

Structural and Functional Characterization of the ABCC6 Transporter in Hepatic Cells: Role on PXE, Cancer Therapy and Drug Resistance.

Pseudoxanthoma elasticum (PXE) is a complex autosomal recessive disease caused by mutations of ABCC6 transporter and characterized by ectopic mineralization of soft connective tissues. Compared to the other ABC transporters, very few studies are available to explain the structural components and working of a full ABCC6 transporter, which may provide some idea about its physiological role in humans. Some studies suggest that mutations of ABCC6 in the liver lead to a decrease in some circulating factor and indicate that PXE is a metabolic disease. It has been reported that ABCC6 mediates the efflux of ATP, which is hydrolyzed in PPi and AMP; in the extracellular milieu, PPi gives potent anti-mineralization effect, whereas AMP is hydrolyzed to Pi and adenosine which affects some cellular properties by modulating the purinergic pathway. Structural and functional studies have demonstrated that silencing or inhibition of ABCC6 with probenecid changed the expression of several genes and proteins such as NT5E and TNAP, as well as Lamin, and CDK1, which are involved in cell motility and cell cycle. Furthermore, a change in cytoskeleton rearrangement and decreased motility of HepG2 cells makes ABCC6 a potential target for anti-cancer therapy. Collectively, these findings suggested that ABCC6 transporter performs functions that modify both the external and internal compartments of the cells.

Relationships between Seminal Plasma Metabolites, Semen Characteristics and Sperm Kinetics in Donkey (Equus asinus).

This study aimed to evaluate donkey seminal plasma metabolites and relate this information to the main characteristics of sperm quality. Sperm kinetics from 10 donkey stallions were analyzed with a computerized system at the time of collection (T0) and after 24 h storage at 4 °C (T24). Seminal plasma was frozen at -80 °C for subsequent proton nuclear magnetic resonance (1H NMR) spectroscopy. On three stallions, semen collection was repeated monthly for three times and sperm analysis also included mitochondrial activity and oxidative status. One stallion was azoospermic and a second semen collection was performed after one month. In the seminal plasma, 17 metabolites were identified; their levels showed numerous significant variations between the azoospermic and the normospermic individuals and grouped in well-defined clusters in a multivariate analysis. Comparing individuals with high and low sperm motility, the only discriminating metabolite was phenylalanine, whose levels were lower in the latter, as in the azoospermic individual. Phenylalanine was also the only metabolite highly correlated with all sperm kinematic parameters at T24. In conclusion, the present study has provided relevant information on the chemical characteristics of donkey semen, identified relationships between seminal metabolites, semen parameters, and sperm kinetics, and offered insights for future technological applications.

Muscari comosum L. Bulb Extracts Modulate Oxidative Stress and Redox Signaling in HepG2 Cells.

Muscari comosum L. bulbs are commonly used as food in South Italy and also in folk medicine. By evaluating in vitro antioxidant activity and biological activities of their aqueous and methanol extracts, we shed light on the potential role, including both the nutraceutical and health benefits, of this plant. Total polyphenol content (TPC) and total flavonoid content (TFC) were evaluated by the Folin-Ciocalteu method and by the aluminum chloride method, respectively. Antioxidant activity was investigated by three in vitro assays and relative antioxidant capacity index (RACI) was calculated to compare results obtained by different tests. The extracts were tested to evaluate their possible involvement in redox homeostasis, using the human hepatoma (HepG2) cell line used as model. The extracts exhibited concentration/solvent dependent radical scavenging activity, as well as dysregulation of some genes involved in redox pathways by promoting Nrf2, SOD-2, GPX1, ABCC6 and ABCG2 expression. NMR metabolomics analysis suggests that HepG2 cells treated with Muscari comosum extracts experience changes in some metabolites involved in various metabolic pathways.

Metabolic changes in follicular fluids of patients treated with recombinant versus urinary human chorionic gonadotropin for triggering ovulation in assisted reproductive technologies: a metabolomics pilot study.

INTRODUCTION: The main goal of this retrospective cohort study is the assessment of the effects of administration of recombinant-hCG (r-hCG) versus urinary-hCG (u-hCG) on follicular fluid (FF) composition of women who underwent in vitro fertilization (IVF) treatments. MATERIALS AND METHODS: We selected 70 patients with infertility attributable to tubal diseases, unexplained infertility, and male factor. Metabolomics analysis of their FFs was performed by 1H nuclear magnetic resonance (1H NMR) spectroscopy in combination with multivariate analysis to interpret the spectral data. Univariate statistical analysis was applied to investigate the possible correlations between clinical parameters and between clinical parameters and metabolites identified by NMR. RESULTS: According to the type of hCG used, significant differences were detected in FFs of women with male factor and unexplained infertility, both in qualitative and quantitative terms, for some metabolites as cholesterol, citrate, creatine, ß-hydroxybutyrate, glycerol, lipids, amino acids (Glu, Gln, His, Val, Lys) and glucose. No significant difference was observed in women with tubal diseases. Besides, the number of MII oocytes in the u-hCG-treated groups correlates positively with glutamate in tubal disease and with glycerol in unexplained infertility. In the r-hCG-treated groups, the number of MII oocytes correlates positively with lipid in tubal disease, positively with citrate and negatively with glucose in male infertility. CONCLUSIONS: Metabolite composition of FF changes according to different type of hCG treatment and this can be related to oocyte development and subsequent outcome. According to the data of this study, different types of hCG should be used in relation to the diagnosis of infertility to obtain better results in inducing oocyte maturation in women undergoing IVF.

Transcriptional Regulation Factors of the Human Mitochondrial Aspartate/Glutamate Carrier Gene, Isoform 2 (SLC25A13): USF1 as Basal Factor and FOXA2 as Activator in Liver Cells.

Mitochondrial carriers catalyse the translocation of numerous metabolites across the inner mitochondrial membrane, playing a key role in different cell functions. For this reason, mitochondrial carrier gene expression needs tight regulation. The human SLC25A13 gene, encoding for the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), catalyses the electrogenic exchange of aspartate for glutamate plus a proton, thus taking part in many metabolic processes including the malate-aspartate shuttle. By the luciferase (LUC) activity of promoter deletion constructs we identified the putative promoter region, comprising the proximal promoter (-442 bp/-19 bp), as well as an enhancer region (-968 bp/-768 bp). Furthermore, with different approaches, such as in silico promoter analysis, gene silencing and chromatin immunoprecipitation, we identified two transcription factors responsible for SLC25A13 transcriptional regulation: FOXA2 and USF1. USF1 acts as a positive transcription factor which binds to the basal promoter thus ensuring SLC25A13 gene expression in a wide range of tissues. The role of FOXA2 is different, working as an activator in hepatic cells. As a tumour suppressor, FOXA2 could be responsible for SLC25A13 high expression levels in liver and its downregulation in hepatocellular carcinoma (HCC).

NMR metabolic profiling of follicular fluid for investigating the different causes of female infertility: a pilot study.

INTRODUCTION: Several metabolomics studies have correlated follicular fluid (FF) metabolite composition with oocyte competence to fertilization, embryo development and pregnancy but there is a scarcity of research examining the metabolic effects of various gynaecological diseases. OBJECTIVES: In this study we aimed to analyze and correlate the metabolic profile of FF from women who were following in vitro fertilization (IVF) treatments with their different infertility pathologies. METHODS: We selected 53 women undergoing IVF who were affected by: tubal diseases, unexplained infertility, endometriosis, polycystic ovary syndrome (PCOS). FF of the study participants was collected at the time of oocytes retrieval. Metabolomic analysis of FF was performed by nuclear magnetic resonance (NMR) spectroscopy. RESULTS: FF presents some significant differences in various infertility pathologies. Although it was not possible to discriminate between FF of control participants and women with tubal diseases and unexplained infertility, comparison of FF metabolic profile from control women with patients with endometriosis and PCOS revealed significant differences in some metabolites that can be correlated to the causes of infertility. CONCLUSION: NMR-based metabolic profiling may be successfully applied to find diagnostic biomarkers for PCOS and endometriosis and it might be also used to predict oocyte developmental potential and subsequent outcome.

Structural characterization of the L0 cytoplasmic loop of human multidrug resistance protein 6 (MRP6).

ABCC6 is a member of the C subfamily of ATP-binding cassette transporters whose mutations are correlated to Pseudoxanthoma elasticum, an autosomal recessive, progressive disorder characterized by ectopic mineralization and fragmentation of elastic fibers. Structural studies of the entire protein have been hindered by its large size, membrane association, and domain complexity. Studies previously performed have contributed to shed light on the structure and function of the nucleotide binding domains and of the N-terminal region. Here we report the expression in E. coli of the polypeptide E205-G279 contained in the cytoplasmic L0 loop. For the first time structural studies in solution were performed. Far-UV CD spectra showed that L0 is structured, assuming predominantly α-helix in TFE solution and turns in phosphate buffer. Fluorescence spectra indicated some flexibility of the regions containing aromatic residues. 1H NMR spectroscopy identified three helical regions separated by more flexible regions.

NMR metabolomics study of follicular fluid in women with cancer resorting to fertility preservation.

PURPOSE: The purpose of this study was to evaluate the possible application of metabolomics to identify follicular fluid changes in cancer patients undergoing fertility preservation. Although metabolomics have been applied already in cancer studies, this is the first application on follicular fluid of cancer patients. METHODS: We selected for the study ten patients with breast cancer and lymphoma who resorted to oocyte cryopreservation to preserve fertility and ten healthy women undergoing in vitro fertilization treatments. Follicular fluid was collected at the time of oocytes retrieval. Metabolomic analysis of follicular fluids was performed by 1H-nuclear magnetic resonance (NMR) spectroscopy in combination with multivariate analysis to interpret the spectral data. Univariate statistical analysis was applied to find correlations between patients' features and metabolites identified by NMR. RESULTS: Partial least squares discriminant analysis allowed to discriminate samples from cancer patients and healthy controls. Univariate statistical analysis found significant correlations between patients' features and metabolites identified by NMR. This finding allowed to identify biomarkers to differentiate both healthy controls from cancer patients and the two different classes of oncological patients. CONCLUSION: The follicular fluids of cancer patients display significant metabolic alterations in comparison to healthy subjects. NMR-based metabolomics could be a valid prognostic tool for identifying and selecting the best cryopreserved oocytes and improving the outcome prediction in cancer women undergoing in vitro fertilization.

Expression, purification and structural characterization of up-regulated gene 7 encoded protein.

Up-Regulated Gene 7 (URG7) is a host gene up-regulated in HBV infected hepatocytes that has been suggested to have an anti-apoptotic activity mediated by caspases 3 and 8 and an endoplasmic reticulum localization. Here we report the structural characterization of the encoded protein URG7 by circular dichroism and fluorescence spectroscopy in different solvent media: phosphate buffer and two membrane-mimetic solvents, i.e. 2,2,2-trifluoroethanol (TFE) and SDS micelles. In all solvents URG7 contains substantial amounts of secondary structures. To obtain information about the structural organization and stability of URG7, its thermal denaturation in a membrane environment was studied and intermediate states of thermal unfolding were observed. Furthermore, fluorescence results in SDS micelles could be compatible with different environments for the four tryptophan residues in URG7. Preliminary NMR data indicate that URG7 in TFE solution is quite flexible and not well folded. These data are the first structural information on URG7 and might provide an insight into its structure-function relationships.

Interaction of cisplatin with a CCHC zinc finger motif.

The interaction between cisplatin and an 18-residue CCHC zinc finger motif derived from a retroviral nucleocapsid protein (PyrZf18) has been studied using UV-visible, CD and (1)H NMR spectroscopies and ESI-MS spectrometry. Cisplatin irreversibly blocks the cysteine zinc binding groups in the free peptide and is able to slowly eject zinc from the zinc-peptide complex. The observed end product of the reaction with cisplatin is a complex in which only one ammonia molecule is coordinated to platinum. After an initial binding with two cysteine residues and the formation of the (PyrZf18)-platinum-(NH3)2 complex, a release of one ammonia molecule occurs because of trans-labilization, and the third cysteine is coordinated, leading to a mixture of isomers and/or conformers of the (PyrZf18)-platinum-NH3 complex. The results are discussed with respect to the potential antiretroviral activity of platinum(II) compounds and to the possible interaction of cisplatin with the cellular nucleic acid binding proteins.

Chemical profile of white wines produced from 'Greco bianco' grape variety in different Italian areas by nuclear magnetic resonance (NMR) and conventional physicochemical analyses.

In this study the characterization of white wines produced from the monovarietal 'Greco bianco' grape variety is presented for the first time. A total of 40 commercial wines, from two different southern Italian regions, Calabria and Campania, from the same grape variety and two different vintages, were investigated. The analyses were performed by means of chromatographic methods, conventional analyses, and nuclear magnetic resonance (NMR) spectroscopy. No differentiation was observed according to the year of production but a significant discrimination was achieved using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). In particular, PLS-DA allowed the selection of compounds (total acidity; citric, malic, succinic, and lactic acids; total polyphenol index; glucose and proline/arginine ratio) useful for differentiating the studied wines on the basis of geographical origin.

The nucleotide-binding domain 2 of the human transporter protein MRP6.

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) belongs to the ABC transporter family, whose members share many characteristic features including membrane domains and two nucleotide-binding domains (NBD1 and NBD2). These function cooperatively to bind and hydrolyze ATP for the transport of substrates across biological membranes. In this study, MRP6-NBD2 (residues 1252-1503) was expressed in Escherichia coli, purified and structurally and functionally characterized. CD spectra suggested that the protein is folded. Furthermore, NBD2 is shown to be biologically active as it binds ATP and presents ATPase activity although significantly lower compared with isolated NBD1. The mixture of NBD2 and NBD1 exhibited an activity similar to the NBD2 alone, indicating that NBD1 and NBD2 form a heterodimer with the latter limiting ATP hydrolysis. These findings suggest that NBD1 has a higher tendency to form an active homodimer, which is also supported by in silico analysis of energy-minimized dimers of the homology models of the two domains.

Study of the nucleotide-binding domain 1 of the human transporter protein MRP6.

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family. Proteins in this family share many characteristic structural features, including two membrane-spanning domains and two nucleotide-binding domains (NBD1 and NBD2), that function cooperatively but not equally bind and hydrolyze ATP. The MRP6 protein is structurally and functionally poorly characterized. Previously, we showed, by NMR spectroscopy, that a fragment of MRP6-NBD1 presents helical structure and fluorescence experiments demonstrated that peptide binds ATP. These data suggested that the study on selected regions could be a valid approach to define the structure of MRP6 . In the present study, to better characterize MRP6-NBD1, we report data of CD spectroscopy, nucleotide binding and ATP hydrolysis on two different polypeptides, one corresponding to the full-length NBD1 domain (residues from Asp-627 to Leu-851) and the other is a shorter polypeptide (residues from Arg-648 to Thr-805) without some key residues. We report that both polypeptides are highly structured in aqueous buffer and in 20% trifluoroethanol showing considerable helical and ß-structure content. The ATP hydrolysis activity is exhibited only by the full-length NBD1 domain. Comparison between our findings and the structurally well characterized MRP1-NBD1 supports the role of H-loop for the ATP hydrolysis and of A-loop in stabilizing the ATP binding.

Biochemical characterization and NMR study of the region E748-A785 of the human protein MRP6/ABCC6.

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family which couple ATP hydrolysis with the transport of molecules across biological membranes. MRP6 topology presents three transmembrane domains and two nucleotide-binding domains (NBDs). The protein is structurally and functionally poorly characterized. Mutations in ABCC6 gene cause Pseudoxanthoma elasticum, a recessive genetic disorder affecting the elastic tissues. Most mutations have been found in NBDs that are critical for ATP binding and hydrolysis. With the aim to better characterize MRP6, we have performed a preliminary study on the fragment E748-A785 of MRP6-NBD1, with the wild type sequence and the R765Q mutation found in PXE affected patients. CD and NMR spectroscopy show the presence of helical structures in both peptides. Fluorescence experiments demonstrate that peptides bind ATP. The NMR structure of the mutated peptide is compared with the corresponding region of the MRP6-NBD1 modeled structure using as a template the X-ray structure of MRP1-NBD1. The finding that both wild type and mutated peptide present the same structure and similar affinity for ATP suggests that the onset of PXE symptoms is a consequence of the different type of interactions involving residue 765 R/Q inside the protein.

Identification of a New Splice Variant of the Human ABCC6 Transporter.

ABCC6 is a member of the adenosine triphosphate-binding cassette (ABC) gene subfamily C that encodes a protein (MRP6) involved in active transport of intracellular compounds to the extracellular environment. Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), an autosomal recessive disorder of the connective tissue characterized by progressive calcification of elastic structures in the skin, the eyes, and the cardiovascular system. MRP6 is codified by 31 exons and contains 1503 amino acids. In addition to a full-length transcript of ABCC6, we have identified an alternatively spliced variant of ABCC6 from a cDNA of human liver that lacks exons 19 and 24. The novel isoform was named ABCC6 Δ19Δ24. PCR analysis from cDNA of cell cultures of primary human hepatocites and embryonic kidney confirms the presence of the ABCC6Δ19Δ24 isoform. Western blot analysis of the embryonic kidney cells shows a band corresponding to the molecular weight of the truncated protein.

Structural characterization of the transmembrane segments of the mitochondrial oxoglutarate carrier (OGC) by NMR spectroscopy.

The oxoglutarate carrier (OGC) is a member of the mitochondrial carrier protein superfamily, which includes the ADP/ATP carrier and other functionally characterized members, and exchanges cytosolic malate for 2-oxoglutarate from the mitochondrial matrix. By means of CD and NMR spectroscopy, we previously characterized four synthetic peptides containing transmembrane segments (TMSs) I, II, V and VI of the OGC, respectively, in TFE/water mixtures and SDS micelles. Here, we present data on the remaining transmembrane segments of OGC obtained by performing CD and NMR studies on peptides corresponding to TMS-III and TMS-IV. In TFE/water, alpha-helical structures were found for these peptides in the L121-R146 and T187-S201 regions, respectively. We also evaluated the compatibility between the helical structures of our peptides and a homology model of the OGC based on the available X-ray structure of the ATP/ADP carrier.