NMR-based metabolomics in giant cell arteritis and polymyalgia rheumatica sequential sera differentiates active and inactive disease.

OBJECTIVES: Giant Cell Arteritis-(GCA) is an inflammatory disease following a chronic, relapsing course. The metabolic alterations related to the intense inflammatory process during the active phase and to the rapid impact of steroid treatment, remain unknown. The study aims to investigate the serum metabolome in active and inactive disease state. METHODS: 110 serum samples from 50 patients [33-GCA and 17-Polymyalgia rheumatica-(PMR)] at 3 time points, 0-(V1: active disease), 1 and 6 months-(V2 and V3: remission) of treatment with glucocorticosteroids (GCs), were subjected to Nuclear Magnetic Resonance (NMR)-based metabolomic analysis. Multi- and univariate statistical analyses were utilized to unveil metabolome alterations following treatment. RESULTS: Distinct metabolic profiles were identified between activity and remission, independently to disease type. N-acetylglycoproteins and cholines of bound phospholipids, emerged as predictive markers of disease activity. Altered levels of 4 out of the 21 small molecules were also observed, including increased levels of phenylalanine, and decreased of glutamine, alanine, and creatinine in active disease. Metabolic fingerprinting discriminated GCA from PMR in remission. GCA and PMR patients exhibited characteristic lipid alterations as a response and/or adverse effect of GCs treatment. Correlation analysis showed that several identified biomarkers were further associated with acute phase reactants, C-Reactive Protein and Erythrocyte Sedimentation Rate. CONCLUSION: The NMR profile of serum metabolome could identify and propose sensitive biomarkers of inflammation. Metabolome alterations, following GCs treatment, could provide predictors for future steroid-induced side effects.

NMR-Based Metabolite Profiling and the Application of STOCSY toward the Quality and Authentication Assessment of European EVOOs.

Extra virgin olive oil (EVOO) possesses a high-value rank in the food industry, thus making it a common target for adulteration. Hence, several methods have been essentially made available over the years. However, the issue of authentication remains unresolved with national and food safety organizations globally struggling to regulate and control its market. Over the course of this study, the aim was to determine the origin of EVOOs suggesting a high-throughput, state-of-the-art method that could be easily adopted. A rapid, NMR-based untargeted metabolite profiling method was applied and complemented by multivariate analysis (MVA) and statistical total correlation spectroscopy (STOCSY). STOCSY is a valuable statistical tool contributing to the biomarker identification process and was employed for the first time in EVOO analysis. Market samples from three Mediterranean countries of Spain, Italy, and Greece, blended samples from these countries, as well as monocultivar samples from Greece were analyzed. The NMR spectra were collected, with the help of chemometrics acting as "fingerprints" leading to the discovery of certain chemical classes and single biomarkers that were related to the classification of the samples into groups based on their origin.

Epigallocatechin gallate is a potent inhibitor of cystathionine beta-synthase: Structure-activity relationship and mechanism of action.

Epigallocatechin gallate (EGCG) is the main bioactive component of green tea. Through screening of a small library of natural compounds, we discovered that EGCG inhibits cystathionine ß-synthase (CBS), a major H2S-generating enzyme. Here we characterize EGCG's mechanism of action in the context of CBS-derived H2S production. In the current project, biochemical, pharmacological and cell biology approaches were used to characterize the effect of EGCG on CBS in cellular models of cancer and Down syndrome (DS). The results show that EGCG binds to CBS and inhibits H2S-producing CBS activity almost 30-times more efficiently than the canonical cystathionine formation (IC50 0.12 versus 3.3 µM). Through screening structural analogs and building blocks, we identified that gallate moiety of EGCG represents the pharmacophore responsible for CBS inhibition. EGCG is a mixed-mode, CBS-specific inhibitor with no effect on the other two major enzymatic sources of H2S, CSE and 3-MST. Unlike the prototypical CBS inhibitor aminooxyacetate, EGCG does not bind the catalytic cofactor of CBS pyridoxal-5'-phosphate. Molecular modeling suggests that EGCG blocks a substrate access channel to pyridoxal-5'-phosphate. EGCG inhibits cellular H2S production in HCT-116 colon cancer cells and in DS fibroblasts. It also exerts effects that are consistent with the functional role of CBS in these cells: in HCT-116 cells it decreases, while in DS cells it improves viability and proliferation. In conclusion, EGCG is a potent inhibitor of CBS-derived H2S production. This effect may contribute to its pharmacological effects in various pathophysiological conditions.

From sample preparation to NMR-based metabolic profiling in food commodities: The case of table olives.

INTRODUCTION: Nuclear magnetic resonance (NMR)-based metabolic profiling has been widely used in food and plant sciences. Despite its simplicity and inherent reproducibility, the determination of the appropriate pre-processing procedures greatly affects the obtained metabolic profile. OBJECTIVES: The current study represents a detailed guide of use for untargeted NMR-based metabolic profiling of table olives (Olea europaea L.). METHODS: Greek Kalamon table olives from different geographical origins were selected as reference materials. Differently treated samples were extracted using different solvents and/or solvent systems. Chemical profiles were evaluated with high-performance thin layer chromatography (HPTLC). Different deuterated solvents and sample concentrations were evaluated for the recording of optimal quality spectra. RESULTS: The methanol extract of freeze-dried table olives was found to contain the most representative secondary metabolites, in higher concentrations, as well. The optimal deuterated solvent for the NMR analysis was methanol-d4 , while final sample concentration should be within the range of 10 to 15 mg/mL. Multivariate data analysis was also used to estimate and confirm the variation and clustering caused by different characteristics of the samples. CONCLUSIONS: Results of the present study make evident the necessity for thorough planning and method development prior to any extensive metabolomic study based on NMR spectroscopy. Pre-processing and sample preparation stages seemed to greatly affect the metabolic profile and spectral quality in the case of table olives, which by extrapolation could apply to other food commodities. Nevertheless, the nature of the samples must be fully described in general, in order to proceed to solid conclusions.

Specificity profile of NAT/NCS2 purine transporters in Sinorhizobium (Ensifer) meliloti.

Sinorhizobium (Ensifer) meliloti is a model example of a soil alpha-proteobacterium which induces the formation of nitrogen-fixing symbiotic nodules on the legume roots. In contrast to all other rhizobacterial species, S. meliloti contains multiple homologs of nucleobase transporter genes that belong to NAT/NCS2 family (Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2). We analyzed functionally all (six) relevant homologs of S. meliloti 1,021 using Escherichia coli K-12 as a host and found that five of them are high-affinity transporters for xanthine (SmLL9), uric acid (SmLL8, SmLL9, SmX28), adenine (SmVC3, SmYE1), guanine (SmVC3), or hypoxanthine (SmVC3). Detailed analysis of substrate profiles showed that two of these transporters display enlarged specificity (SmLL9, SmVC3). SmLL9 is closely related in sequence with the xanthine-specific XanQ of E. coli. We subjected SmLL9 to rationally designed site-directed mutagenesis and found that the role of key binding-site residues of XanQ is conserved in SmLL9, whereas a single amino-acid change (S93N) converts the xanthine/uric-acid transporter SmLL9 to a xanthine-preferring variant, due to disruption of an essential hydrogen bond with the C8 oxygen of uric acid. The results highlight the presence of several different purine nucleobase transporters in S. meliloti and imply that the purine transport might be important in the nodule symbiosis involving S. meliloti.

Exploring the metabolomic profile of cerebellum after exposure to acute stress.

Psychological stress and stress-related disorders constitute a major health problem in modern societies. Although the brain circuits involved in emotional processing are intensively studied, little is known about the implication of cerebellum in stress responses whereas the molecular changes induced by stress exposure in cerebellum remain largely unexplored. Here, we investigated the effects of acute stress exposure on mouse cerebellum. We used a forced swim test (FST) paradigm as an acute stressor. We then analyzed the cerebellar metabolomic profiles of stressed (n = 11) versus control (n = 11) male CD1 mice by a Nuclear Magnetic Resonance (NMR)-based, untargeted metabolomics approach. Our results showed altered levels of 19 out of the 47 annotated metabolites, which are implicated in neurotransmission and N-acetylaspartic acid (NAA) turnover, as well as in energy and purine/pyrimidine metabolism. We also correlated individual metabolite levels with FST behavioral parameters, and reported associations between FST readouts and levels of 4 metabolites. This work indicates an altered metabolomic signature after acute stress in the cerebellum and highlights a previously unexplored involvement of cerebellum in stress responses.

Design, synthesis, and biological evaluation of new raloxifene analogues of improved antagonist activity and endometrial safety.

Raloxifene agonism of estrogen receptor (ER) in post-menopausal endometrium is not negligible. Based on a rational drug design workflow, we synthesized 14 analogues of raloxifene bearing a polar group in the aromatic ring of the basic side chain (BSC) and/or changes in the bulkiness of the BSC amino group. Analogues with a polar BSC aromatic ring and amino group substituents of increasing volume displayed increasing ER antagonism in Ishikawa cells. Analogues with cyclohexylaminoethoxy (13a) or adamantylaminoethoxy BSC (13b) lacking a polar aromatic ring displayed high ER-binding affinity and ER antagonism in Ishikawa cells higher than raloxifene and similar to fulvestrant (ICI182,780). The endometrial surface epithelium of immature female CD1 mice injected with 13b was comparable to that of vehicle-treated mice, while that of mice treated with estradiol, raloxifene or 13b in combination with estradiol was hyperplastic. These findings indicate that raloxifene analogues with a bulky BSC amino group could provide for higher endometrial safety treatment of the menopausal syndrome.

The effect of prolonged intense physical exercise of special forces volunteers on their plasma protein denaturation profile examined by differential scanning calorimetry.

The human blood plasma proteome profile has been an area of intensive investigation and differential scanning calorimetry (DSC) has come forward as a novel tool in analyzing plasma heat capacity changes to monitor various physiological responses in health and disease. This study used DSC to assess potential alterations in the plasma heat capacity profile of albumin and globulins during extremely demanding physical exercise. We monitored the changes in denaturation profiles of those plasma proteins for five consecutive days of an extraordinary exercise training schedule in 14 young male Special Forces volunteers, as well as after a 30-day recovery period. The major effect of the prolonged intense exercise was the continuous upward shift of the albumin peak by 2°-3 °C on the initial days of exercise, with a tendency to plateau circa the 5th day of exercise. In addition, some redistribution of the denaturational enthalpy was observed upon exercise, where the globulins peak increased relative to the albumin peak. Noteworthy, the alterations in the plasma proteome denaturational profiles were not persistent, as virtually full recovery of the initial status was observed after 30 days of recovery. Our findings indicate that 5 days of exhaustive physical exercise of highly trained individuals enhanced the thermal stability of plasma albumin shifting its denaturational transition to higher temperatures. We surmise that these effects may be a result of increased blood oxygenation during the prolonged intense exercise and, consequently, of albumin oxidation as part of the overall adaptation mechanisms of the body to extreme physical and/or oxidative stress.

Glycyrrhiza glabra-Enhanced Extract and Adriamycin Antiproliferative Effect on PC-3 Prostate Cancer Cells.

Prostate cancer is the second most commonly diagnosed cancer in men worldwide, which is almost incurable, once it progresses into the metastatic stage. Adriamycin (ADR) is a known chemotherapeutic agent that causes severe side effects. In recent years, studies in natural plant products have revealed their anticancer activities. In particular, Glycyrrhiza glabra enhanced extract (GGE), commonly known as licorice, has been reported to exert antiproliferative properties against cancer cells. In this study, the cytotoxic potential of GGE was assessed in PC-3 cells, when it is administrated alone or in combination with Adriamycin. PC-3 cells were treated with GGE and/or ADR, and the inhibition of cell proliferation was evaluated by the MTT assay. Cell cycle alterations and apoptosis rate were measured through flow cytometry. Expression levels of autophagy-related genes were evaluated with specific ELISA kits, Western blotting, and real-time PCR, while NMR spectrometry was used to identify the implication of specific metabolites. Our results demonstrated that GGE alone or in co-treatment with ADR shows antiproliferative properties against PC-3 cells, which are mediated by both apoptosis and autophagy mechanisms.

Effects of lifelong exercise and aging on the blood metabolic fingerprint of rats.

Regular exercise is an important part of a healthy lifestyle, as it helps maintain a healthy weight and reduces the risk of chronic diseases. We explored the effects of lifelong exercise and aging on rat metabolism through a metabolomics approach. Thirty-six rats were divided into four equal groups: exercise during the 1st half of life (3-12 months), lifelong exercise (3-21 months), no exercise, and exercise during the 2nd half of life (12-21 months). Exercise consisted in swimming for 20 min, five times a week. Blood samples collected at 3, 12, and 21 months of life were analysed by 1H NMR spectroscopy. The groups that exercised during the 2nd half of life weighed less than the groups that did not. Exercise had an orexigenic effect during the 1st half and an anorexigenic effect during the 2nd half. Multivariate analysis showed a clear discrimination between ages when groups were treated as one and between the exercising and non-exercising groups at 12 months. Univariate analysis showed many effects of aging and some effects of exercise on metabolites involved in carbohydrate, lipid and protein metabolism. Especially during the 1st half, exercise had anabolic effects, whereas aging had catabolic effects on amino acid metabolism. In two cases (glycine and succinate), exercise (especially during the 1st half) mitigated potentially harmful effects of aging. The higher values of succinate and the lower values of lactate during the 1st half in the exercising groups suggest increased oxidative metabolism. In conclusion, moderate-intensity exercise for life or half-life had strong and potentially healthful effects on body weight and (partly) appetite, as well as on some blood metabolites. The effects of aging on the rat blood metabolome seemed to be stronger than those of exercise.

Steroid sulfatase inhibiting lanostane triterpenes - Structure activity relationship and in silico insights.

Steroid sulfatase (STS) transforms hormone precursors into active steroids. Thus, it represents a target of intense research regarding hormone-dependent cancers. In this study, three ligand-based pharmacophore models were developed to identify STS inhibitors from natural sources. In a pharmacophore-based virtual screening of a curated molecular TCM database, lanostane-type triterpenes (LTTs) were predicted as STS ligands. Three traditionally used polypores rich in LTTs, i.e., Ganoderma lucidum Karst., Gloeophyllum odoratum Imazeki, and Fomitopsis pinicola Karst., were selected as starting materials. Based on eighteen thereof isolated LTTs a structure activity relationship for this compound class was established with piptolinic acid D (1), pinicolic acid B (2), and ganoderol A (3) being the most pronounced and first natural product STS inhibitors with IC50 values between 10 and 16 µM. Molecular docking studies proposed crucial ligand target interactions and a prediction tool for these natural compounds correlating with experimental findings.

Human Melanoma-Cell Metabolic Profiling: Identification of Novel Biomarkers Indicating Metastasis.

Melanoma is the most aggressive type of skin cancer, leading to metabolic rewiring and enhancement of metastatic transformation. Efforts to improve its early and accurate diagnosis are largely based on preclinical models and especially cell lines. Hence, we herein present a combinational Nuclear Magnetic Resonance (NMR)- and Ultra High Performance Liquid Chromatography-High-Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS)-mediated untargeted metabolomic profiling of melanoma cells, to landscape metabolic alterations likely controlling metastasis. The cell lines WM115 and WM2664, which belong to the same patient, were examined, with WM115 being derived from a primary, pre-metastatic, tumor and WM2664 clonally expanded from lymph-node metastases. Metabolite samples were analyzed using NMR and UHPLC-HRMS. Multivariate statistical analysis of high resolution NMR and MS (positive and negative ionization) results was performed by Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), while metastasis-related biomarkers were determined on the basis of VIP lists, S-plots and Student's t-tests. Receiver Operating Characteristic (ROC) curves of NMR and MS data revealed significantly differentiated metabolite profiles for each cell line, with WM115 being mainly characterized by upregulated levels of phosphocholine, choline, guanosine and inosine. Interestingly, WM2664 showed notably increased contents of hypoxanthine, myo-inositol, glutamic acid, organic acids, purines, pyrimidines, AMP, ADP, ATP and UDP(s), thus indicating the critical roles of purine, pyrimidine and amino acid metabolism during human melanoma metastasis.

Malignancy Grade-Dependent Mapping of Metabolic Landscapes in Human Urothelial Bladder Cancer: Identification of Novel, Diagnostic, and Druggable Biomarkers.

BACKGROUND: Urothelial bladder cancer (UBC) is one of the cancers with the highest mortality rate and prevalence worldwide; however, the clinical management of the disease remains challenging. Metabolomics has emerged as a powerful tool with beneficial applications in cancer biology and thus can provide new insights on the underlying mechanisms of UBC progression and/or reveal novel diagnostic and therapeutic schemes. METHODS: A collection of four human UBC cell lines that critically reflect the different malignancy grades of UBC was employed; RT4 (grade I), RT112 (grade II), T24 (grade III), and TCCSUP (grade IV). They were examined using Nuclear Magnetic Resonance, Mass Spectrometry, and advanced statistical approaches, with the goal of creating new metabolic profiles that are mechanistically associated with UBC progression toward metastasis. RESULTS: Distinct metabolic profiles were observed for each cell line group, with T24 (grade III) cells exhibiting the most abundant metabolite contents. AMP and creatine phosphate were highly increased in the T24 cell line compared to the RT4 (grade I) cell line, indicating the major energetic transformation to which UBC cells are being subjected during metastasis. Thymosin ß4 and ß10 were also profiled with grade-specific patterns of expression, strongly suggesting the importance of actin-cytoskeleton dynamics for UBC advancement to metastatic and drug-tolerant forms. CONCLUSIONS: The present study unveils a novel and putatively druggable metabolic signature that holds strong promise for early diagnosis and the successful chemotherapy of UBC disease.

NMR-Based Metabolic Profiling of Edible Olives-Determination of Quality Parameters.

Edible olive drupes (from Olea europaea L.) are a high-value food commodity with an increasing production trend over the past two decades. In an attempt to prevent fraud issues and ensure quality, the International Olive Council (IOC) issued guidelines for their sensory evaluation. However, certain varieties, geographical origins and processing parameters are omitted. The aim of the present study was the development of a method for the quality assessment of edible olives from the Konservolia, Kalamon and Chalkidikis cultivars from different areas of Greece processed with the Spanish or Greek method. A rapid NMR-based untargeted metabolic profiling method was developed along with multivariate analysis (MVA) and applied for the first time in edible olives' analysis complemented by the aid of statistical total correlation spectroscopy (STOCSY). Specific biomarkers, related to the classification of olives based on different treatments, cultivars and geographical origin, were identified. STOCSY proved to be a valuable aid towards the assignment of biomarkers, a bottleneck in untargeted metabolomic approaches.

Screening of Heteroaromatic Scaffolds against Cystathionine Beta-Synthase Enables Identification of Substituted Pyrazolo[3,4-c]Pyridines as Potent and Selective Orthosteric Inhibitors.

Cystathionine ß-synthase (CBS) is a key enzyme in the production of the signaling molecule hydrogen sulfide, deregulation of which is known to contribute to a range of serious pathological states. Involvement of hydrogen sulfide in pathways of paramount importance for cellular homeostasis renders CBS a promising drug target. An in-house focused library of heteroaromatic compounds was screened for CBS modulators by the methylene blue assay and a pyrazolopyridine derivative with a promising CBS inhibitory potential was discovered. The compound activity was readily comparable to the most potent CBS inhibitor currently known, aminoacetic acid, while a promising specificity over the related cystathionine γ-lyase was identified. To rule out any possibility that the inhibitor may bind the enzyme regulatory domain due to its high structural similarity with cofactor s-adenosylmethionine, differential scanning fluorimetry was employed. A sub-scaffold search guided follow-up screening of related compounds, providing preliminary structure-activity relationships with respect to requisites for efficient CBS inhibition by this group of heterocycles. Subsequently, a hypothesis regarding the exact binding mode of the inhibitor was devised on the basis of the available structure-activity relationships (SAR) and a deep neural networks analysis and further supported by induced-fit docking calculations.

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli.

The uracil permease UraA of Escherichia coli is a structurally known prototype for the ubiquitous Nucleobase-Ascorbate Transporter (NAT) or Nucleobase-Cation Symporter-2 (NCS2) family and represents a well-defined subgroup of bacterial homologs that remain functionally unstudied. Here, we analyze four of these homologs, including RutG of E. coli which shares 35% identity with UraA and is encoded in the catabolic rut (pyrimidine utilization) operon. Using amplified expression in E. coli K-12, we show that RutG is a high-affinity permease for uracil, thymine and, at low efficiency, xanthine and recognizes also 5-fluorouracil and oxypurinol. In contrast, UraA and the homologs from Acinetobacter calcoaceticus and Aeromonas veronii are permeases specific for uracil and 5-fluorouracil. Molecular docking indicates that thymine is hindered from binding to UraA by a highly conserved Phe residue which is absent in RutG. Site-directed replacement of this Phe with Ala in the three uracil-specific homologs allows high-affinity recognition and/or transport of thymine, emulating the RutG profile. Furthermore, all RutG orthologs from enterobacteria retain an Ala at this position, implying that they can use both uracil and thymine and, possibly, xanthine as substrates and provide the bacterial cell with a range of catabolizable nucleobases.

Indirubin Analogues Inhibit Trypanosoma brucei Glycogen Synthase Kinase 3 Short and T. brucei Growth.

The protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). The disease is fatal if it remains untreated, whereas most drug treatments are inadequate due to high toxicity, difficulties in administration, and low central nervous system penetration. T. brucei glycogen synthase kinase 3 short (TbGSK3s) is essential for parasite survival and thus represents a potential drug target that could be exploited for HAT treatment. Indirubins, effective leishmanicidals, provide a versatile scaffold for the development of potent GSK3 inhibitors. Herein, we report on the screening of 69 indirubin analogues against T. brucei bloodstream forms. Of these, 32 compounds had potent antitrypanosomal activity (half-maximal effective concentration = 0.050 to 3.2 µM) and good selectivity for the analogues over human HepG2 cells (range, 7.4- to over 641-fold). The majority of analogues were potent inhibitors of TbGSK3s, and correlation studies for an indirubin subset, namely, the 6-bromosubstituted 3'-oxime bearing an extra bulky substituent on the 3' oxime [(6-BIO-3'-bulky)-substituted indirubins], revealed a positive correlation between kinase inhibition and antitrypanosomal activity. Insights into this indirubin-TbGSK3s interaction were provided by structure-activity relationship studies. Comparison between 6-BIO-3'-bulky-substituted indirubin-treated parasites and parasites silenced for TbGSK3s by RNA interference suggested that the above-described compounds may target TbGSK3s in vivo To further understand the molecular basis of the growth arrest brought about by the inhibition or ablation of TbGSK3s, we investigated the intracellular localization of TbGSK3s. TbGSK3s was present in cytoskeletal structures, including the flagellum and basal body area. Overall, these results give insights into the mode of action of 6-BIO-3'-bulky-substituted indirubins that are promising hits for antitrypanosomal drug discovery.

Silymarin Enriched Extract (Silybum marianum) Additive Effect on Doxorubicin-Mediated Cytotoxicity in PC-3 Prostate Cancer Cells.

Silymarin-enriched extract (SEE) is obtained from Silybum marianum (Asteraceae). Doxorubicin (DXR) is a widely used chemotherapeutical yet with severe side effects. The goal of the present study was to assess the pharmacologic effect of SEE and its bioactive components silibinin and silychristine when administrated alone or in combination with DXR in the human prostate cancer cells (PC-3). PC-3 cells were treated with SEE, silibinin (silybins A and B), silychristine, alone, and in combination with DXR, and cell proliferation was assessed by the MTT assay. Cell cycle, apoptosis, and autophagy rate were assessed by flow cytometry. Expression levels of autophagy-related genes were quantified by qRT-PCR, ELISA and western blot while transmission electron microscopy was performed to reveal autophagic structures. Finally, NMR spectrometry was used to identify specific metabolites related to autophagy. SEE inhibited PC-3 cell proliferation in a dose-dependent manner while the co-treatment (DXR-SEE) revealed an additive cytotoxic effect. Cell cycle, apoptosis, and autophagy variations were observed in addition to altered expression levels of autophagy related genes (LC3, p62, NBR1, Beclin1, ULK1, AMBRA1), while several modifications in autophagic structures were identified after DXR-SEE co-treatment. Furthermore, treated cells showed a different metabolic profile, with significant alterations in autophagy-related metabolites such as branched-chain amino acids. In conclusion, the DXR-SEE co-treatment provokes perturbations in the autophagic mechanism of prostate cancer cells (PC-3) compared to DXR treatment alone, causing an excessive cell death. These findings propose the putative use of SEE as an adjuvant cytotoxic agent.

Cryptic purine transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family.

NCS1 proteins are H+ or Na+ symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are classified into two evolutionary and structurally distinct subfamilies, known as Fur- and Fcy-like transporters. These subfamilies have expanded and functionally diversified by gene duplications. The Fur subfamily of the model fungus Aspergillus nidulans includes both major and cryptic transporters specific for uracil, 5-fluorouracil, allantoin or/and uric acid. Here we functionally analyse all four A. nidulans Fcy transporters (FcyA, FcyC, FcyD and FcyE) with previously unknown function. Our analysis shows that FcyD is moderate-affinity, low-capacity, highly specific adenine transporter, whereas FcyE contributes to 8-azaguanine uptake. Mutational analysis of FcyD, supported by homology modelling and substrate docking, shows that two variably conserved residues (Leu356 and Ser359) in transmembrane segment 8 (TMS8) are critical for transport kinetics and specificity differences among Fcy transporters, while two conserved residues (Phe167 and Ser171) in TMS3 are also important for function. Importantly, mutation S359N converts FcyD to a promiscuous nucleobase transporter capable of recognizing adenine, xanthine and several nucleobase analogues. Our results reveal the importance of specific residues in the functional evolution of NCS1 transporters.

NmeA, a novel efflux transporter specific for nucleobases and nucleosides, contributes to metal resistance in Aspergillus nidulans.

Through Minos transposon mutagenesis we obtained A. nidulans mutants resistant to 5-fluorouracil due to insertions into the upstream region of the uncharacterized gene nmeA, encoding a Major Facilitator Superfamily (MFS) transporter. Minos transpositions increased nmeA transcription, which is otherwise extremely low under all conditions tested. To dissect the function of NmeA we used strains overexpressing or genetically lacking the nmeA gene. Strains overexpressing NmeA are resistant to toxic purine analogues, but also, to cadmium, zinc and borate, whereas an isogenic nmeAΔ null mutant exhibits increased sensitivity to these compounds. We provide direct evidence that nmeA overexpression leads to efflux of adenine, xanthine, uric acid and allantoin, the latter two being intermediate metabolites of purine catabolism that are toxic when accumulated cytoplasmically due to relevant genetic lesions. By using a functional GFP-tagged version we show that NmeA is a plasma membrane transporter. Homology modeling and docking approaches identified a single purine binding site and a tentative substrate translocation trajectory in NmeA. Orthologues of NmeA are present in all Aspergilli and other Eurotiomycetes, but are absent from other fungi or non-fungal organisms. NmeA is thus the founding member of a new class of transporters essential for fungal success under specific toxic conditions.