BBA - General Subjects (v.1850, #1)

A central role for intermolecular dityrosine cross-linking of fibrinogen in high molecular weight advanced oxidation protein product (AOPP) formation by Graziano Colombo; Marco Clerici; Daniela Giustarini; Nicola Portinaro; Salvatore Badalamenti; Ranieri Rossi; Aldo Milzani; Isabella Dalle-Donne (1-12).
Advanced oxidation protein products (AOPPs) are dityrosine cross-linked and carbonyl-containing protein products formed by the reaction of plasma proteins with chlorinated oxidants, such as hypochlorous acid (HOCl). Most studies consider human serum albumin (HSA) as the main protein responsible for AOPP formation, although the molecular composition of AOPPs has not yet been elucidated. Here, we investigated the relative contribution of HSA and fibrinogen to generation of AOPPs.AOPP formation was explored by SDS-PAGE, under both reducing and non-reducing conditions, as well as by analytical gel filtration HPLC coupled to fluorescence detection to determine dityrosine and pentosidine formation.Following exposure to different concentrations of HOCl, HSA resulted to be carbonylated but did not form dityrosine cross-linked high molecular weight aggregates. Differently, incubation of fibrinogen or HSA/fibrinogen mixtures with HOCl at concentrations higher than 150 μM induced the formation of pentosidine and high molecular weight (HMW)-AOPPs (> 200 kDa), resulting from intermolecular dityrosine cross-linking. Dityrosine fluorescence increased in parallel with increasing HMW-AOPP formation and increasing fibrinogen concentration in HSA/fibrinogen mixtures exposed to HOCl. This conclusion is corroborated by experiments where dityrosine fluorescence was measured in HOCl-treated human plasma samples containing physiological or supra-physiological fibrinogen concentrations or selectively depleted of fibrinogen, which highlighted that fibrinogen is responsible for the highest fluorescence from dityrosine.A central role for intermolecular dityrosine cross-linking of fibrinogen in HMW-AOPP formation is shown.These results highlight that oxidized fibrinogen, instead of HSA, is the key protein for intermolecular dityrosine formation in human plasma.
Keywords: Advanced oxidation protein products; Albumin; Fibrinogen; Hypochlorous acid; Human plasma; Dityrosine;

Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis by Matías D. Asención Diez; Ana M. Demonte; Karl Syson; Diego G. Arias; Andrii Gorelik; Sergio A. Guerrero; Stephen Bornemann; Alberto A. Iglesias (13-21).
Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected.In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation.Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism.This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen.
Keywords: ADP-glucose pyrophosphorylase; Glycogen synthase; UDP-glucose pyrophosphorylase; Trehalose-6-phosphate synthase; Phosphoenolpyruvate; Glucose-6-phosphate;

Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress.hiPS cells were seeded on a Matrigel-coated dish and cultured to induce differentiation into a dopamine neuronal linage. On day 18 of culture, cells were collected from the culture dishes and seeded into U-bottom 96-well plates to induce cell aggregate formation. After 5 days, cell aggregates were collected from the plates and microencapsulated in agarose microbeads. The microencapsulated aggregates were cultured for an additional 45 days to induce maturation of dopamine neurons.Approximately 60% of all cells differentiated into tyrosine hydroxylase-positive neurons in agarose microbeads. The cells released dopamine for more than 40 days. In addition, microbeads containing cells could be cryopreserved.hiPS cells were successfully differentiated into dopamine neurons in agarose microbeads.Agarose microencapsulation provides a good supporting environment for the preparation and storage of dopamine neurons.
Keywords: Induced pluripotent stem cell; Dopamine neuron; Microencapsulation;

Advances in mass spectrometry driven O-glycoproteomics by Steven B. Levery; Catharina Steentoft; Adnan Halim; Yoshiki Narimatsu; Henrik Clausen; Sergey Y. Vakhrushev (33-42).
Global analyses of proteins and their modifications by mass spectrometry are essential tools in cell biology and biomedical research. Analyses of glycoproteins represent particular challenges and we are only at the beginnings of the glycoproteomic era. Some of the challenges have been overcome with N-glycoproteins and proteome-wide analysis of N-glycosylation sites is accomplishable today but only by sacrificing information of structures at individual glycosites. More recently advances in analysis of O-glycoproteins have been made and proteome-wide analysis of O-glycosylation sites is becoming available as well.Here we discuss the challenges of analysis of O-glycans and new O-glycoproteomics strategies focusing on O-GalNAc and O-Man glycoproteomes.A variety of strategies are now available for proteome-wide analysis of O-glycosylation sites enabling functional studies. However, further developments are still needed for complete analysis of glycan structures at individual sites for both N- and O-glycoproteomics strategies.The advances in O-glycoproteomics have led to identification of new biological functions of O-glycosylation and a new understanding of the importance of where O-glycans are positioned on proteins.
Keywords: Orbitrap; ETD; SimpleCell; Glycopeptide; Lectin; Site-specific;

It is well-known that elevated amounts of nitric oxide and other reactive nitrogen species (RNS) impact negatively on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. These perturbations severely compromise O2-dependent energy production. While bacteria are known to adapt to RNS, a key tool employed by macrophages to combat infections, the exact mechanisms are unknown.The bacterium was cultured in a defined mineral medium and cell-free extracts obtained at the same growth phase were utilized for various biochemical studies Blue native polyacrylamide gel electrophoresis followed by in-gel activity assays, high performance liquid chromatography and co-immunoprecipitaton are applied to investigate the effects of RNS on the model microbe Pseudomonas fluorescens.Citrate is channeled away from the tricarboxylic acid cycle using a novel metabolon consisting of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK). This metabolic engine comprising three disparate enzymes appears to transiently assemble as a supercomplex aimed at ATP synthesis. The up-regulation in the activities of adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK) ensured the efficacy of this ATP-making machine.Microbes may escape the effects of nitrosative stress by re-engineering metabolic networks in order to generate and store ATP anaerobically when the electron transport chain is defective.The molecular configuration described herein provides further understanding of how metabolism plays a key role in the adaptation to nitrosative stress and reveals novel targets that will inform the development of antimicrobial agents to counter RNS-resistant pathogens.Display Omitted
Keywords: Metabolon; Reactive nitrogen species; Phosphotransfer; Metabolism; Microbiology;

The mechanism of action of ursolic acid as insulin secretagogue and insulinomimetic is mediated by cross-talk between calcium and kinases to regulate glucose balance by Allisson Jhonatan Gomes Castro; Marisa Jádna Silva Frederico; Luisa Helena Cazarolli; Camila Pires Mendes; Lizandra Czermainski Bretanha; Éder Carlos Schmidt; Zenilda Laurita Bouzon; Veronica Aiceles de Medeiros Pinto; Cristiane da Fonte Ramos; Moacir Geraldo Pizzolatti; Fátima Regina Mena Barreto Silva (51-61).
The effect of in vivo treatment with ursolic acid (UA) on glycemia in hyperglycemic rats and its mechanism of action on muscle were studied.The UA effects on glycemia, glycogen, LDH, calcium and on insulin levels were evaluated after glucose tolerance curve. The β-cells were evaluated through the transmission electron microscopy. UA mechanism of action was studied on muscles through the glucose uptake with/without specific insulin signaling inhibitors. The nuclear effect of UA and the GLUT4 expression on muscle were studied using thymidine, GLUT4 immunocontent, immunofluorescence and RT-PCR.UA presented a potent antihyperglycemic effect, increased insulin vesicle translocation, insulin secretion and augmented glycogen content. Also, UA stimulates the glucose uptake through the involvement of the classical insulin signaling related to the GLUT4 translocation to the plasma membrane as well as the GLUT4 synthesis. These were characterized by increasing the GLUT4 mRNA expression, the activation of DNA transcription, the expression of GLUT4 and its presence at plasma membrane. Also, the modulation of calcium, phospholipase C, protein kinase C and PKCaM II is mandatory for the full stimulatory effect of UA on glucose uptake. UA did not change the serum LDH and serum calcium balance.The antihyperglycemic role of UA is mediated through insulin secretion and insulinomimetic effect on glucose uptake, synthesis and translocation of GLUT4 by a mechanism of cross-talk between calcium and protein kinases.UA is a potential anti-diabetic agent with pharmacological properties for insulin resistance and diabetes therapy.
Keywords: Calcium; Secretagogue; GLUT4; Hyperglycemia; Insulin; Triterpene;

L312, a novel PPARγ ligand with potent anti-diabetic activity by selective regulation by Xinni Xie; Xinbo Zhou; Wei Chen; Long Long; Wei Li; Xiuyan Yang; Song Li; Lili Wang (62-72).
Selective PPARγ modulators (sPPARγM) retains insulin sensitizing activity but with minimal side effects compared to traditional TZDs agents, is thought as a promising strategy for development of safer insulin sensitizer.We used a combination of virtual docking, SPR-based binding, luciferase reporter and adipogenesis assays to analyze the interaction mode, affinity and agonistic activity of L312 to PPARγ in vitro, respectively. And the anti-diabetic effects and underlying molecular mechanisms of L312 was studied in db/db mice.L312 interacted with PPARγ-LBD in a manner similar to known sPPARγM. L312 showed similar PPARγ binding affinity, but displayed partial PPARγ agonistic activity compared to PPARγ full agonist pioglitazone. In addition, L312 displayed partial recruitment of coactivator CBP yet equal disassociation of corepressor NCoR1 compared to pioglitazone. In db/db mice, L312 (30 mg/kg·day) treatment considerably improved insulin resistance with the regard to OGTT, ITT, fasted blood glucose, HOMA-IR and serum lipids, but elicited less weight gain, adipogenesis and hemodilution compared with pioglitazone. Further studies demonstrated that L312 is a potent inhibitor of CDK5-mediated PPARγ phosphorylation and displayed a selective gene expression profile in epididymal WAT.L312 is a novel sPPARγM.L312 may represent a novel lead for designing ideal sPPARγM for T2DM treatment with advantages over current TZDs.
Keywords: Selective PPARγ modulator; pSer273PPARγ; Insulin resistance;

A new erythrocyte-based biochemical approach to predict the antiproliferative effects of heterocyclic scaffolds: The case of indolone by Angela Scala; Silvana Ficarra; Annamaria Russo; Davide Barreca; Elena Giunta; Antonio Galtieri; Giovanni Grassi; Ester Tellone (73-79).
The indole core is a key structural feature of many natural products and biomolecules with broad spectrum chemotherapeutic properties. Some of us have recently synthesized a library of biologically promising indolone-based compounds. The present study focuses on the effects of one of them, namely DPIT, on human erythrocytes.We have examined the influence of DPIT on band 3 protein, intracellular ATP concentration and transport, caspase 3 activation, metabolic adaptation and membrane stability.Our study elucidates that DPIT, intercalated into the phospholipid bilayer, decreases the anion transport, the intracellular ATP concentration and the cytosolic pH, inducing a direct activation of caspase 3.Starting from the metabolic similarity between erythrocytes and cancer cells, we investigate how the metabolic derangements and membrane alterations induced by selected heterocycles could be related to the antiproliferative effects.Our work aims to propose a new model of study to predict the antiproliferative effects of heterocyclic scaffolds, pointing out that only one of the listed conditions would be unfavorable to the life cycle of neoplastic cells.Display Omitted
Keywords: Heterocycle; Red blood cell; Anion transport; Caspase 3; ATP transport; Antiproliferation;

Magainin-AM2 improves glucose homeostasis and beta cell function in high-fat fed mice by O.O. Ojo; D.K. Srinivasan; B.O. Owolabi; J.M. Conlon; P.R. Flatt; Y.H.A. Abdel-Wahab (80-87).
Magainin-AM2, a previously described amphibian host-defense peptide, stimulates insulin- and glucagon-like peptide-1-release in vitro. This study investigated anti-diabetic effects of the peptide in mice with diet-induced obesity and glucose intolerance.Male National Institute of Health Swiss mice were maintained on a high-fat diet for 12-weeks prior to the daily treatment with magainin-AM2. Various indices of glucose tolerance were monitored together with insulin secretory responsiveness of islets at conclusion of study.Following twice daily treatment with magainin-AM2 for 15 days, no significant difference in body weight and food intake was observed compared with saline-treated high fat control animals. However, non-fasting blood glucose was significantly (P < 0.05) decreased while plasma insulin concentrations were significantly (P < 0.05) increased. Oral and intraperitoneal glucose tolerance and insulin secretion following glucose administration via both routes were significantly (P < 0.05) enhanced. The peptide significantly (P < 0.001) improved insulin sensitivity as well as the beta cell responses of islets isolated from treated mice to a range of insulin secretagogues. Oxygen consumption, CO2 production, respiratory exchange ratio and energy expenditure were not significantly altered by sub-chronic administration of magainin-AM2 but a significant (P < 0.05) reduction in fat deposition was observed.These results indicate that magainin-AM2 improves glucose tolerance, insulin sensitivity and islet beta cells secretory responsiveness in mice with obesity-diabetes.The activity of magainin-AM2 suggests the possibility of exploiting this peptide for treatment of type 2 diabetes.
Keywords: Magainin-AM2; Amphibian peptide; Type 2 diabetes; Obesity; Insulin-release;

The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate by Ana C. Ebrecht; Matías D. Asención Diez; Claudia V. Piattoni; Sergio A. Guerrero; Alberto A. Iglesias (88-96).
Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes.Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification.Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides.The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.
Keywords: UDP-glucose pyrophosphorylase; Redox regulated; Glucose-1-phosphate; Galactose-1-phosphate; Carbohydrate metabolism;

The North American wood frog, Rana sylvatica, is able to overcome subzero conditions through overwintering in a frozen state. Freezing imposes ischemic and oxidative stress on cells as a result of cessation of blood flow. Superoxide dismutases (SODs) catalyze the redox reaction involving the dismutation of superoxide (O2 −• ) to molecular oxygen and hydrogen peroxide.The present study investigated the regulation of CuZnSOD and MnSOD kinetics as well as the transcript, protein and phosphorylation levels of purified enzyme from the muscle of control and frozen R. sylvatica.CuZnSOD from frozen muscle showed a significantly higher Vmax (1.52 fold) in comparison to CuZnSOD from the muscle of control frogs. MnSOD from frozen muscle showed a significantly lower Km for O2 −• (0.66 fold) in comparison to CuZnSOD from control frogs. MnSOD from frozen frogs showed higher phosphorylation of serine (2.36 fold) and tyrosine (1.27 fold) residues in comparison to MnSOD from control animals. Susceptibility to digestion via thermolysin after incubation with increasing amount of urea (Cm) was tested, resulting in no significant changes for CuZnSOD, whereas a significant change in MnSOD stability was observed between control (2.53 M urea) and frozen (2.92 M urea) frogs. Expressions of CuZnSOD and MnSOD were quantified at both mRNA and protein levels in frog muscle, but were not significantly different.The physiological consequence of freeze-induced SOD modification appears to adjust SOD function in freezing frogs.Augmented SOD activity may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.
Keywords: Rana sylvatica; Freeze tolerance; Ischemia; Oxidative stress; Antioxidant; Protein stability;

Mitochondrial proteases act on STARD3 to activate progesterone synthesis in human syncytiotrophoblast by Mercedes Esparza-Perusquía; Sofía Olvera-Sánchez; Oscar Flores-Herrera; Héctor Flores-Herrera; Alberto Guevara-Flores; Juan Pablo Pardo; María Teresa Espinosa-García; Federico Martínez (107-117).
STARD1 transports cholesterol into mitochondria of acutely regulated steroidogenic tissue. It has been suggested that STARD3 transports cholesterol in the human placenta, which does not express STARD1. STARD1 is proteolytically activated into a 30-kDa protein. However, the role of proteases in STARD3 modification in the human placenta has not been studied.Progesterone determination and Western blot using anti-STARD3 antibodies showed that mitochondrial proteases cleave STARD3 into a 28-kDa fragment that stimulates progesterone synthesis in isolated syncytiotrophoblast mitochondria. Protease inhibitors decrease STARD3 transformation and steroidogenesis.STARD3 remained tightly bound to isolated syncytiotrophoblast mitochondria. Simultaneous to the increase in progesterone synthesis, STARD3 was proteolytically processed into four proteins, of which a 28-kDa protein was the most abundant. This protein stimulated mitochondrial progesterone production similarly to truncated-STARD3. Maximum levels of protease activity were observed at pH 7.5 and were sensitive to 1,10-phenanthroline, which inhibited steroidogenesis and STARD3 proteolytic cleavage. Addition of 22(R)-hydroxycholesterol increased progesterone synthesis, even in the presence of 1,10-phenanthroline, suggesting that proteolytic products might be involved in mitochondrial cholesterol transport.Metalloproteases from human placental mitochondria are involved in steroidogenesis through the proteolytic activation of STARD3. 1,10-Phenanthroline inhibits STARD3 proteolytic cleavage. The 28-kDa protein and the amino terminal truncated-STARD3 stimulate steroidogenesis in a comparable rate, suggesting that both proteins share similar properties, probably the START domain that is involved in cholesterol binding.Mitochondrial proteases are involved in syncytiotrophoblast-cell steroidogenesis regulation. Understanding STARD3 activation and its role in progesterone synthesis is crucial to getting insight into its action mechanism in healthy and diseased syncytiotrophoblast cells.
Keywords: Human syncytiotrophoblast mitochondria; Progesterone synthesis; STARD3 protein; Mitochondrial metalloprotease;

Potential biological role of laccase from the sponge Suberites domuncula as an antibacterial defense component by Qiang Li; Xiaohong Wang; Michael Korzhev; Heinz C. Schröder; Thorben Link; Muhammad Nawaz Tahir; Bärbel Diehl-Seifert; Werner E.G. Müller (118-128).
Laccases are copper-containing enzymes that catalyze the oxidation of a wide variety of phenolic substrates.We describe the first poriferan laccase from the marine demosponge Suberites domuncula.This enzyme comprises three characteristic multicopper oxidase homologous domains. Immunohistological studies revealed that the highest expression of the laccase is in the surface zone of the animals. The expression level of the laccase gene is strongly upregulated after exposure of the animals to the bacterial endotoxin lipopolysaccharide. To allow the binding of the recombinant enzyme to ferromagnetic nanoparticles, a recombinant laccase was prepared which contained in addition to the His-tag, a Glu-tag at the N-terminus of the enzyme. The recombinant laccase was enzymatically active. The apparent Michaelis constant of the enzyme is 114 μM, using syringaldazine as substrate. Exposure of E. coli to the nanoparticles, coated with Glu-tagged laccase, and to the mediator 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in the presence of lignin, as the oxidizable substrate, resulted in an almost complete inhibition of colony formation. Quantitative studies of the effect of the laccase-coated iron oxide nanoparticles were performed using E. coli grown in suspension in reaction tubes within a magnetic nanoparticle separator.This newly designed magnetic nanoparticle separator allowed a removal of the nanoparticles after terminating the reaction. Using this system, a strong dose-dependent inhibition of the growth of E. coli by the laccase iron oxide nanoparticles was determined.From our data we conclude that the sponge laccase is involved in the anti-bacterial defense of the sponge organism.A siliceous sponge (Suberites domuncula) comprises a laccase [Lac] that functions as a radical generating enzyme by which bacteria are killed. In order to enhance the function the enzyme employs a mediator, e.g. ABTS.Display Omitted
Keywords: Laccase; Copper; Sponges; Anti-bacterial defense; Lignin; Ferromagnetic particles;

A dihydroindolizino indole derivative selectively stabilizes G-quadruplex DNA and down-regulates c-MYC expression in human cancer cells by Narayana Nagesh; G. Raju; R. Srinivas; P. Ramesh; M. Damoder Reddy; Ch. Raji Reddy (129-140).
Telomeric and NHE III1, a c-MYC promoter region is abundant in guanine content and readily form G-quadruplex structures. Small molecules that stabilize G-quadruplex DNA were shown to reduce oncoprotein expression, initiate apoptosis and they may function as anticancer molecules.Electrospray ionization mass spectrometry, spectroscopy, isothermal titration calorimetry, Taq DNA polymerase stop assay, real time PCR and luciferase reporter assay. Cell migration assay to find out the effect of derivatives on normal as well as cancer cell proliferation.Among three different dihydroindolizino indole derivatives, 4-cyanophenyl group attached derivative has shown maximum affinity, selective interaction and higher stability towards G-quadruplex DNA over dsDNA. Further, as a potential G-quadruplex DNA stabilizer, 4-cyanophenyl linked dihydroindolizino indole derivative was found to be more efficient in inhibiting in vitro DNA synthesis, c-MYC expression and cancer cell proliferation among human cancer cells.The present study reveals that dihydroindolizino indole derivative having 4-cyanophenyl group has potential to stabilize G-quadruplex DNA and exhibit anticancer activity.These studies are useful in the identification and synthesis of lead derivatives that will selectively stabilize G-quadruplex DNA and function as anticancer agents.A series of dihydroindolizino indole derivatives were synthesized and their antitumor activity, ability to selectively interact and stabilize G-quadruplex DNA were studied. Results indicate that 4-cyanophenyl linked derivative (DHII-4CPh) is superior in all aspects. It is closely followed by thiophen-2-yl (DHII-TPh) and 3-methyl-1-phenyl-1H-pyrazol-4-yl (DHII-MPhPy) linked derivatives.Display Omitted
Keywords: Dihydroindolizino indole; ESI-MS; Spectroscopy; Selective stabilization of G-quadruplex DNA; Reduce c-MYC expression; Anticancer activity;

Methionyl-7-amino-4-methylcoumarin (MetAMC) serves as a substrate for the Escherichia coli methionine aminopeptidase (MetAP) catalyzed reaction, and is routinely used for screening compounds to identify potential antibiotic agents. In pursuit of screening the enzyme's inhibitors, we observed that 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), utilized to solubilize hydrophobic inhibitors, inhibited the catalytic activity of the enzyme, and such inhibition was not solely due to sequestration of the substrate by HP-β-CD.The mechanistic path for the HP-β-CD mediated inhibition of MetAP was probed by performing a detailed account of steady-state kinetics, ligand binding, X-ray crystallographic, and molecular modeling studies.X-ray crystallographic data of the β-cyclodextrin–substrate (β-CD–MetAMC) complex reveal that while the AMC moiety of the substrate is confined within the CD cavity, the methionine moiety protrudes outward. The steady-state kinetic data for inhibition of MetAP by HP-β-CD–MetAMC conform to a model mechanism in which the substrate is “bridged” between HP-β-CD and the enzyme's active-site pocket, forming HP-β-CD–MetAMC–MetAP as the catalytically inactive ternary complex. Molecular modeling shows that the scissile bond of HP-β-CD-bound MetAMC substrate does not reach within the proximity of the enzyme's catalytic metal center, and thus the substrate fails to undergo cleavage.The data presented herein suggests that the bridging of the substrate between the enzyme and HP-β-CD cavities is facilitated by interaction of their surfaces, and the resulting complex inhibits the enzyme activity.Due to its potential interaction with physiological proteins via sequestered substrates, caution must be exercised in HP-β-CD mediated delivery of drugs under pathophysiological conditions.
Keywords: Substrate bridging; Cyclodextrin; Methionine aminopeptidase; Inhibition; Cyclodextrin–substrate complex; Molecular modeling;

In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates by Leszek Wawiórka; Dawid Krokowski; Yuliya Gordiyenko; Daniel Krowarsch; Carol V. Robinson; Ishag Adam; Nikodem Grankowski; Marek Tchórzewski (150-158).
The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins.We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods.All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)2 form represents the most favorable architecture for parasite P-proteins.The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1–P2 heterodimeric intermediate.On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role.
Keywords: Ribosomes; Protein complexes; Protein–protein interactions; Malaria; Ribosomal P proteins; Ribosomal stalk;

A novel synthetic luteinizing hormone-releasing hormone (LHRH) analogue coupled with modified β-cyclodextrin: Insight into its intramolecular interactions by Golfo G. Kordopati; Theodore V. Tselios; Tahsin Kellici; Franci Merzel; Thomas Mavromoustakos; Simona Golic Grdadolnik; Gerasimos M. Tsivgoulis (159-168).
Cyclodextrins (CDs) in combination with therapeutic proteins and other bioactive compounds have been proposed as candidates that show enhanced chemical and enzymatic stability, better absorption, slower plasma clearance and improved dose–response curves or immunogenicity. As a result, an important number of therapeutic complexes between cyclodextrins and bioactive compounds capable to control several diseases have been developed.In this article, the synthesis and the structural study of a conjugate between a luteinizing hormone-releasing hormone (LHRH) analogue, related to the treatment of hormone dependent cancer and fertility, and modified β-cyclodextrin residue are presented. The results show that both the phenyl group of tyrosine (Tyr) as well as the indole group of tryptophan (Trp) can be encapsulated inside the cyclodextrin cavity. Solution NMR experiments provide evidence that these interactions take place intramolecularly and not intermolecularly.The study of a LHRH analogue conjugated with modified β-cyclodextrin via high field NMR and MD experiments revealed the existence of intramolecular interactions that could lead to an improved drug delivery.NMR in combination with MD simulation is of great value for a successful rational design of peptide–cyclodextrin conjugates showing stability against enzymatic proteolysis and a better pharmacological profile.Display Omitted
Keywords: LHRH; Cyclodextrin; NMR spectroscopy; Drug delivery; Intramolecular interaction;

Engineered chimeras reveal the structural basis of hexacoordination in globins: A case study of neuroglobin and myoglobin by Ignacio Boron; Luciana Capece; Francesca Pennacchietti; Diana E. Wetzler; Stefano Bruno; Stefania Abbruzzetti; Lucía Chisari; F. Javier Luque; Cristiano Viappiani; Marcelo A. Marti; Dario A. Estrin; Alejandro D. Nadra (169-177).
Myoglobin (Mb) and neuroglobin (Ngb) are representative members of pentacoordinated and bis-histidyl, hexacoordinated globins. In spite of their low sequence identity, they show surprisingly similar three-dimensional folds. The ability of Ngb to form a hexacoordinated bis-histidyl complex with the distal HisE7 has a strong impact on ligand affinity. The factors governing such different behaviors have not been completely understood yet, even though they are extremely relevant to establish structure–function relationships within the globin superfamily.In this work we generated chimeric proteins by swapping a previously identified regulatory segment – the CD region – and evaluated comparatively the structural and functional properties of the resulting proteins by molecular dynamics simulations, and spectroscopic and kinetic investigations.Our results show that chimeric proteins display heme coordination properties displaced towards those expected for the corresponding CD region. In particular, in the absence of exogenous ligands, chimeric Mb is found as a partially hexacoordinated bis-histidyl species, whereas chimeric Ngb shows a lower equilibrium constant for forming a hexacoordinated bis-histidyl species.While these results confirm the regulatory role of the CD region for bis-histidyl hexacoordination, they also suggest that additional sources contribute to fine tune the equilibrium. General significance Globins constitute a ubiquitous group of heme proteins widely found in all kingdoms of life. These findings raise challenging questions regarding the structure–function relationships in these proteins, as bis-histidyl hexacoordination emerges as a novel regulatory mechanism of the physiological function of globins.Display Omitted
Keywords: Neuroglobin; Myoglobin; Bis-histidyl coordination; Molecular dynamics; Flash photolysis;

Non-alcoholic steatohepatitis (NASH) is a subset of non-alcoholic fatty liver disease, the most common chronic liver disease in the U.S. Fibrosis, a common feature of NASH, results from the dysregulation of fibrogenesis in hepatic stellate cells (HSCs). In this study, we investigated whether astaxanthin (ASTX), a xanthophyll carotenoid, can inhibit fibrogenic effects of transforming growth factor β1 (TGFβ1), a key fibrogenic cytokine, in HSCs.Reactive oxygen species (ROS) accumulation was measured in LX-2, an immortalized human HSC cell line. Quantitative realtime PCR, Western blot, immunocytochemical analysis, and in-cell Western blot were performed to determine mRNA and protein of fibrogenic genes, and the activation of Smad3 in TGFβ1-activated LX-2 cells and primary mouse HSCs.In LX-2 cells, ROS accumulation induced by tert-butyl hydrogen peroxide and TGFβ1 was abolished by ASTX. ASTX significantly decreased TGFβ1-induced α-smooth muscle actin (α-SMA) and procollagen type 1, alpha 1 (Col1A1) mRNA as well as α-SMA protein levels. Knockdown of Smad3 showed the significant role of Smad3 in the expression of α-SMA and Col1A1, but not TGFβ1, in LX-2 cells. ASTX attenuated TGFβ1-induced Smad3 phosphorylation and nuclear translocation with a concomitant inhibition of Smad3, Smad7, TGFβ receptor I (TβRI), and TβRII expression. The inhibitory effect of ASTX on HSC activation was confirmed in primary mouse HSCs as evidenced by decreased mRNA and protein levels of α-SMA during activation.Taken together, ASTX exerted anti-fibrogenic effects by blocking TGFβ1-signaling, consequently inhibiting the activation of Smad3 pathway in HSCs.This study suggests that ASTX may be used as a preventive/therapeutic agent to prevent hepatic fibrosis.
Keywords: Astaxanthin; Non-alcoholic steatohepatitis; Liver fibrosis; Hepatic stellate cell; Transforming growth factor β1; Smad3;

A guide into glycosciences: How chemistry, biochemistry and biology cooperate to crack the sugar code by Dolores Solís; Nicolai V. Bovin; Anthony P. Davis; Jesús Jiménez-Barbero; Antonio Romero; René Roy; Karel Smetana; Hans-Joachim Gabius (186-235).
The most demanding challenge in research on molecular aspects within the flow of biological information is posed by the complex carbohydrates (glycan part of cellular glycoconjugates). How the ‘message’ encoded in carbohydrate ‘letters’ is ‘read’ and ‘translated’ can only be unraveled by interdisciplinary efforts.This review provides a didactic step-by-step survey of the concept of the sugar code and the way strategic combination of experimental approaches characterizes structure–function relationships, with resources for teaching.The unsurpassed coding capacity of glycans is an ideal platform for generating a broad range of molecular ‘messages’. Structural and functional analyses of complex carbohydrates have been made possible by advances in chemical synthesis, rendering production of oligosaccharides, glycoclusters and neoglycoconjugates possible. This availability facilitates to test the glycans as ligands for natural sugar receptors (lectins). Their interaction is a means to turn sugar-encoded information into cellular effects. Glycan/lectin structures and their spatial modes of presentation underlie the exquisite specificity of the endogenous lectins in counterreceptor selection, that is, to home in on certain cellular glycoproteins or glycolipids.Understanding how sugar-encoded ‘messages’ are ‘read’ and ‘translated’ by lectins provides insights into fundamental mechanisms of life, with potential for medical applications.
Keywords: Conformer; Crystallography; Dendrimer; Glycan; Lectin; Neoglycoconjugate;

The mucins found as components of mucus gel layers at mucosal surfaces throughout the body play roles in protection as part of the defensive barrier on an organ and tissue specific basis.The human MUC gene family codes up to 20 known proteins, which can be divided into secreted and membrane-associated forms each with a typical protein domain structure. The secreted mucins are adapted to cross link in order to allow formation of the extended mucin networks found in the secreted mucus gels. The membrane-associated mucins possess membrane specific domains which enable their various biological functions as part of the glycocalyx. All mucins are highly O-glycosylated and this is tissue specific and linked with specific biological functions at these locations. Mucin biology is dynamic and the processes of degradation and turnover are well integrated with biosynthesis to maintain a continuous mucosal protection against all external aggressive forces. Interaction of mucins with microflora plays an important role in normal function. Mucins are modified in a variety of diseases and this may be due to abberant mucin peptide or glycosylation.Mucins represent a family of glycoprotein having fundamental roles in mucosal protection and communication with external environment.The review emphasises the nature of mucins as glycoproteins and their role in presenting an array of glycan structures at the mucosal cell surface.
Keywords: Mucin; Mucus; Glycosylation; Mucosal; Microflora; Cancer;

Retraction notice to “Acetyl-11-keto-β-boswellic acid (AKBA) inhibits human gastric carcinoma growth through modulation of the Wnt/β-catenin signaling pathway” [1830 (6) 3604–3615] by Yi Yuan; Yu-Sheng Zhang; Ji-Zhen Xie; Julia-Li Zhong; Yuan-Yuan Li; Rui-Qi Wang; Yi-Zhuo Qin; Hong-Xiang Lou; Zu-Hua Gao; Xian-Jun Qu (254).