BBA - General Subjects (v.1850, #9)

Regulation and RNA-binding properties of Hfq-like RNA chaperones in Bacillus anthracis by Gurudutta Panda; Pooja Tanwer; Salim Ansari; Devanshi Khare; Rakesh Bhatnagar (1661-1668).
Small RNAs (sRNAs) are important modulators of gene expression in bacteria. Regulation by sRNAs is yet to be established in Bacillus anthracis. Here, regulation and RNA-binding properties of Hfq-like RNA chaperones in B. anthracis are investigated.Transcript levels were measured by RT-PCR. Proteins were cloned, purified, and their ability to bind sRNA was seen by EMSA. Regulators of Hfq1 were identified by in silico analysis and validated by EMSA. Conserved sRNAs were identified by homology search and their ability to bind Hfq1 was seen by EMSA. Residues crucial for sRNA binding were identified by mutational studies. hfq1 and hfq3 showed expression during exponential phase on BHI medium, while hfq2 showed no expression. Hfq1 and Hfq3 formed hexamer and sRNA–Hfq complex, not Hfq2. In silico prediction and EMSA confirmed AbrB binding to the promoter of Hfq1. Homology search identified 7 sRNAs in B. anthracis. The sRNA CsfG showed binding to Hfq1 via residues Y24, F29, Q30, R32, K56, and H57.Hfq1 and Hfq3 can function as RNA chaperones in B. anthracis. The transition phase repressor AbrB might be responsible for the growth-dependent expression of Hfq1. The sporulation-specific sRNA CsfG binds to Hfq1 via its distal surface and requires an intact hexameric structure for forming CsfG-Hfq1 complex.This is the first report demonstrating the regulation and functional properties of Hfq-like RNA chaperones in B. anthracis and paves the path towards establishing sRNA-based regulation in B. anthracis.
Keywords: Hfq; RNA chaperones; Bacillus anthracis; Small RNAs; AbrB; CsfG;

Maturation of human iPS cell-derived dopamine neuron precursors in alginate–Ca2 + hydrogel by Mitsue Komatsu; Shuhei Konagaya; Edgar Y. Egawa; Hiroo Iwata (1669-1675).
Pluripotent stem cells (embryonic stem/induced pluripotent stem cells) have been widely studied as a potential cell source for cell transplantation therapy of Parkinson's disease. However, some difficulties remain to be overcome. These include the need to prepare a large number of dopamine (DA) neurons for clinical use and to culture the cells for a long period to allow their functional maturation and the removal of undifferentiated cells.In this study, aggregates of DA neuron precursors were enclosed in alginate–Ca2 + microbeads, and the encapsulated aggregates were cultured for 25 days to induce cell maturation.More than 60% of cells in the aggregates differentiated into tyrosine hydroxylase-positive DA neurons. The aggregates could release DA at the same level as aggregates maintained on culture dishes without encapsulation. In addition, by exposure to a citrate solution, the alginate–Ca2 + gel layer could be easily removed from aggregates without damaging the DA neurons. When the aggregates were transplanted into rat brain, viable cells were found in the graft at one week post-transplantation, with cells extending neurites into the host tissue.Cell aggregates encapsulated in alginate–Ca2 + beads successfully differentiated into mature DA neurons.The alginate–Ca2 + microbead is suitable for maintaining DA precursor aggregates for a long period to allow their functional maturation.
Keywords: iPS cell; Dopamine; Neuron precursor; Alginate; Microencapsulation;

A novel anti-microtubule agent with carbazole and benzohydrazide structures suppresses tumor cell growth in vivo by Makoto Ohira; Yuka Iwasaki; Chika Tanaka; Michitaka Kuroki; Naoki Matsuo; Tatsuhiko Kitamura; Masaki Yukuhiro; Hiroyuki Morimoto; Nisha Pang; Bei Liu; Tohru Kiyono; Masahide Amemiya; Kozo Tanaka; Kazumasa Yoshida; Nozomi Sugimoto; Takashi Ohshima; Masatoshi Fujita (1676-1684).
The mitotic spindles are among the most successful targets of anti-cancer chemotherapy, and they still hold promise as targets for novel drugs. The anti-mitotic drugs in current clinical use, including taxanes, epothilones, vinca alkaloids, and halichondrins, are all microtubule-targeting agents. Although these drugs are effective for cancer chemotherapy, they have some critical problems; e.g., neurotoxicity caused by damage to neuronal microtubules, as well as innate or acquired drug resistance. To overcome these problems, a great deal of effort has been expended on development of novel anti-mitotics.We identified novel microtubule-targeting agents with carbazole and benzohydrazide structures: N′-[(9-ethyl-9H-carbazol-3-yl)methylene]-2-methylbenzohydrazide (code number HND-007) and its related compounds. We investigated their activities against cancer cells using various methods including cell growth assay, immunofluorescence analysis, cell cycle analysis, tubulin polymerization assay, and tumor inhibition assay in nude mice.HND-007 inhibits tubulin polymerization in vitro and blocks microtubule formation and centrosome separation in cancer cells. Consequently, it suppresses the growth of various cancer cell lines, with IC50 values in the range 1.3–4.6 μM. In addition, HND-007 can inhibit the growth of taxane-resistant cancer cells that overexpress P-glycoprotein. Finally, HND-007 can inhibit HeLa cell tumor growth in nude mice.Taken together, these findings suggest that HND-007 is a promising lead compound for development of novel anti-mitotic, anti-microtubule chemotherapeutic agents.Display Omitted
Keywords: Anti-mitotic agents; Anti-neoplastic agents; Centrosome; Microtubules; Mitotic spindle;

Post-translational modification of a chimeric EPO-Fc hormone is more important than its molecular size in defining its in vivo hematopoietic activity by Emilio R. Salgado; Raquel Montesino; Sivana P. Jiménez; Mauricio González; Florence Hugues; Oscar I. Cabezas; Rafael Maura-Perez; Paulina Saavedra; Emilio Lamazares; Alexis Salas-Burgos; Juan C. Vera; Oliberto Sánchez; Jorge R. Toledo (1685-1693).
Recombinant erythropoietin (EPO) has been marketed as biopharmaceutical for anemia and chronic renal failure. Long-acting EPO variants that aimed at achieving less frequent dosing have been generated, either by the addition of glycosylation sites or increasing its molecular weight.The hEPO cDNA linked to the human IgG Fc fragment was cloned as a single codifying gene on the pAdtrack-CMV vector, yielding the recombinant adenoviral genome. For in vitro and in vivo expression assays cervical cancer cell line (SiHa) and nulliparous goats were used, respectively. The hematopoietic activity of EPO-Fc, expressed as the differential increment of hematocrit was evaluated in B6D2F1 mice. NP-HPLC of the 2AB-labeled N-glycan was carried out to profile analysis.The direct transduction of mammary secretory cells with adenoviral vector is a robust methodology to obtain high levels of EPO of up to 3.5 mg/mL in goat's milk. SiHa-derived EPO-Fc showed significant improvement in hematopoietic activity compared to the commercial hEPO counterpart or with the homologous milk-derived EPO-Fc. The role of the molecular weight seemed to be important in enhancing the hematopoietic activity of SiHa-derived EPO-Fc. However, the lack of sialylated multi-antennary glycosylation profile in milk-derived EPO-Fc resulted in lower biological activity.The low content of tri- or tetra-antennary sialylated N-glycans linked to the chimeric EPO-Fc hormone, expressed in the goat mammary gland epithelial cells, defined its in vivo hematopoietic activity.The sialylated N-glycan content plays a more significant role in the in vivo biological activity of hEPO than its increased molecular weight.Display Omitted
Keywords: Erythropoietin; Adenovirus vector; N-glycans; Mammary epithelial cell; Milk expression; Hematopoietic activity;

Mistic-fused expression of algal rhodopsins in Escherichia coli and its photochemical properties by Keon Ah Lee; Sang-Soo Lee; So Young Kim; Ah Reum Choi; Jung-Ha Lee; Kwang-Hwan Jung (1694-1703).
Since algal rhodopsins, the eukaryotic seven-transmembrane proteins, are generally difficult to express in Escherichia coli, eukaryotic cells have been used for heterologous expression. Mistic, a membrane-associated protein that was originally discovered in Bacillus subtilis, has been shown to improve the expression levels of many foreign integral membrane proteins in E. coli when used as a fusion partner linked to the N-terminus of cargo proteins.Here, we expressed two algal rhodopsins with N- and C-terminal Mistic domains in E. coliAcetabularia rhodopsin I (ARI) and Chlamydomonas sensory rhodopsin B (CSRB, channel rhodopsin 2). UV/VIS spectroscopy, pH titration of proton acceptor residue, laser-induced photolysis and electrophysiological measurement were used for investigating important residues in proton transport and spectroscopic characters of the proteins.Protein yield of two algal rhodopsins was enhanced, obtaining 0.12 mg of Mistic-ARI and 0.04 mg of Mistic-CSRB per liter of culture. Spheroplast expression Mistic-ARI had outward proton-pumping activity, indicating protein functionality. Asp89 of ARI changed its protonation state by light absorption, and Asp100 was important for O600 formation. Electrophysiology revealed that both residues took part in proton transport. The spectroscopic analyses of Mistic-CSRB revealed its characteristics.Fusion to the membrane-integrating protein Mistic can enhance overexpression of eukaryotic type I rhodopsins in E. coli.These findings indicate that Mistic fusion and E. coli expression method could be an effective, low cost technique for studying eukaryotic membrane proteins. This may have useful implications, for example, in studying structural characteristics and optogenetics for rhodopsins.
Keywords: Rhodopsin; Bacteriorhodopsin; Algal rhodopsin; Proton transfer; Ion channel;

A comprehensive glycome profiling of Huntington's disease transgenic mice by Solomon T. Gizaw; Toshiaki Koda; Maho Amano; Keiko Kamimura; Tetsu Ohashi; Hiroshi Hinou; Shin-Ichiro Nishimura (1704-1718).
Huntington's disease (HD) is an autosomal, dominantly inherited and progressive neurodegenerative disease, nosologically classified as the presence of intranuclear inclusion bodies and the loss of GABA-containing neurons in the neostriatum and subsequently in the cerebellar cortex. Abnormal processing of neuronal proteins can result in the misfolding of proteins and altered post-translational modification of newly synthesized proteins. Total glycomics, namely, N-glycomics, O-glycomics, and glycosphingolipidomics (GSL-omics) of HD transgenic mice would be a hallmark for central nervous system disorders in order to discover disease specific biomarkers.Glycoblotting method, a high throughput glycomic protocol, and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) were used to study the total glycome expression levels in the brain tissue (3 mice of each sex) and sera (5 mice of each sex) of HD transgenic and control mice. All experiments were performed twice and differences in the expression levels of major glycoforms were compared between HD transgenic and control mice.We estimated the structure and expression levels of 87 and 58  N-glycans in brain tissue and sera, respectively, of HD transgenic and control mice. The present results clearly indicated that the brain glycome and their expression levels are significantly gender specific when compared with those of other tissues and serum. Core-fucosylated and bisecting-GlcNAc types of N-glycans were found in increased levels in the brain tissue HD transgenic mice. Accordingly, core-fucosylated and sialic acid (particularly N-glycolylneuraminic acid, NeuGc) for biantennary type glycans were found in increased amounts in the sera of HD transgenic mice compared to that of control mice. Core 3 type O-glycans were found in increased levels in male and in decreased levels in both the striatum and cortexes of female HD transgenic mice. Furthermore, serum levels of core 1 type O-glycans decreased and were undetected for core 2 type O-glycans for HD transgenic mice. In glycosphingolipids, GD1a in brain tissue and GM2-NeuGc serum levels were found to have increased and decreased, respectively, in HD transgenic mice compared to those of the control group mice.Total glycome expression levels are significantly different between HD transgenic and control group mice.Glycoblotting combined with MALDI-TOF/MS total glycomics warrants a comprehensive, effective, novel and versatile technique for qualitative and quantitative analysis of total glycome expression levels. Furthermore, glycome-focused studies of both environmentally and genetically rooted neurodegenerative diseases are promising candidates for the discovery of potential disease glyco-biomarkers in the post-genome era.Display Omitted
Keywords: Huntington's disease; Total glycomics; Transgenic mouse; Glycoblotting; Glyco-biomarker;

Biochemical, structural and functional diversity between two digestive α-amylases from Helicoverpa armigera by Amey J. Bhide; Sonal M. Channale; Sucheta S. Patil; Vidya S. Gupta; Sureshkumar Ramasamy; Ashok P. Giri (1719-1728).
Helicoverpa armigera (Lepidoptera) feeds on various plants using diverse digestive enzymes as one of the survival tool-kit. The aim of the present study was to understand biochemical properties of recombinant α-amylases of H. armigera viz., HaAmy1 and HaAmy2.The open reading frames of HaAmy1 and HaAmy2 were cloned in Pichia pastoris and expressed heterologously. Purified recombinant enzymes were characterized for their biochemical and biophysical attributes using established methods.Sequence alignment and homology modeling showed that HaAmy1 and HaAmy2 were conserved in their amino acid sequences and structures. HaAmy1 and HaAmy2 showed optimum activity at 60 °C; however, they differed in their optimum pH. Furthermore, HaAmy2 showed higher affinity for starch and amylopectin whereas HaAmy1 had higher catalytic efficiency. HaAmy1 and HaAmy2 were inhibited to the same magnitude by a synthetic amylase inhibitor (acarbose) while wheat amylase inhibitor showed about 2-fold higher inhibition of HaAmy1 than HaAmy2 at pH 7 while 6-fold difference at pH 11. Interactions of HaAmy1 and HaAmy2 with wheat amylase inhibitor revealed 2:1 stoichiometric ratio and much more complex interaction with HaAmy1.The diversity of amylases in perspective of their biochemical and biophysical properties, and their differential interactions with amylase inhibitors signify the potential role of these enzymes in adaptation of H. armigera on diverse plant diets.Characterization of digestive enzymes of H. armigera provides the molecular basis for the polyphagous nature and thus could assist in designing future strategies for the insect control.Display Omitted
Keywords: Helicoverpa armigera; α-amylases; Digestive enzymes; Enzyme activity; Amylase inhibitors;

Oleic acid complex of bovine α-lactalbumin induces eryptosis in human and other erythrocytes by a Ca2 +-independent mechanism by Mehboob Hoque; Ravikanth Nanduri; Jyoti Gupta; Sahil Mahajan; Pawan Gupta; M. Saleemuddin (1729-1739).
Complexes of oleic acid (OA) with milk α-lactalbumin, received remarkable attention in view of their selective toxicity towards a spectrum of tumors during the last two decades. OA complexes of some structurally related/unrelated proteins are also tumoricidal. Erythrocytes are among the few differentiated cells that are sensitive and undergo hemolysis when exposed to the complexes.The effects of OA complex of bovine α-lactalbumin (Bovine Alpha-lactalbumin Made LEthal to Tumor cells, BAMLET) on human, goat and chicken erythrocytes on calcein leakage, phosphatidylserine exposure, morphological changes and hemolysis were studied by confocal microscopy, FACS analysis, scanning electron microscopy and measuring hemoglobin release.Erythrocytes exposed to BAMLET undergo eryptosis-like alterations as revealed by calcein leakage, surface phosphatidylserine exposure and transformation to echinocytes at low concentrations and hemolysis when the concentration of the complex was raised. Ca2 + was not essential and restricted the alterations when included in the medium. The BAMLET-induced alterations in human erythrocytes were prevented by the cation channel inhibitors, amiloride and BaCl2 but not by inhibitors of thiol proteases, sphingomyelinase and by the antioxidant N-acetyl cysteine.The work shows for the first time that low concentrations of BAMLET induces eryptosis in erythrocytes by a novel mechanism not requiring Ca2 + and hemolysis by detergent-like action by the released OA at higher concentrations.The study points out to the need for a comprehensive evaluation of the toxicity of OA complexes of α-lactalbumin and other proteins towards erythrocytes and other differentiated cells before being considered for therapy.
Keywords: HAMLET/BAMLET; Eryptosis; Oleic acid; Antitumor drugs; α-Lactalbumin;

While apocrine secretion was among the earliest secretory mechanisms to be identified, its underlying basis remains poorly understood.This review reappraises our understanding of apocrine secretion using insights about apocrine secretion from the salivary glands of Drosophila, in which molecular genetic analyses have provided a glimmer of hope for elucidating the mechanistic aspects of this fundamental process.In contrast to the well-defined process of exocytosis, apocrine secretion is non-vesicular transport and secretory pathway that entails the loss of part of the cytoplasm. It often involves apical protrusions and generates cytoplasmic fragments inside a secretory lumen. In its most intense phase this process is accompanied by the release of large fragments of cellular structures and entire organelles that include mitochondria, Golgi, and portions of the endoplasmic reticulum, among others. Proteomic analyses revealed that the secretion is composed of hundreds to thousands of membranous, cytoskeletal, microsomal, mitochondrial, ribosomal, and even nuclear as well as nucleolar proteins. Strikingly, although many nuclear proteins are released, the nuclear deoxyribonucleic acid itself remains intact. In spite of this complexity, it appears that several protein components of apocrine secretion are identical, regardless of the location of the apocrine gland.This type of secretion appears to be common to many, if not all, barrier epithelial tissues including skin derivatives and the epididymis, and is implicated also in lung/bronchi and intestinal epithelium. Apocrine secretion is a mechanism that provides the en masse delivery of a very complex proteinaceous mixture from polarized epithelial tissues to allow for communication at exterior interfaces.Display Omitted
Keywords: Apocrine secretion; Noncanonical traffic mechanism; Apocrine glands; Secretory components; Proteomics; Evolutionary origin of secretion;

Geraniin exerts cytoprotective effect against cellular oxidative stress by upregulation of Nrf2-mediated antioxidant enzyme expression via PI3K/AKT and ERK1/2 pathway by Peng Wang; Xiao Peng; Zuo-Fu Wei; Fu-Yao Wei; Wei Wang; Wei-Dong Ma; Li-Ping Yao; Yu-Jie Fu; Yuan-Gang Zu (1751-1761).
Geraniin, an active compound with remarkable antioxidant activity, was isolated from Geranium sibiricum. The present study aimed to investigate whether geraniin has the ability to activate Nrf2, induce antioxidant enzyme expression and protect cells from oxidative damage.The cells were pretreated with geraniin for 24 h and exposed to hydrogen peroxide (H2O2) for 4 h. Intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential and apoptosis were measured. We also investigated intracellular glutathione (GSH) levels and changes in nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling cascade in cells treated with geraniin.We investigated the protective effects of geraniin against H2O2-induced apoptosis in HepG2 cells. Geraniin significantly reduced H2O2-induced oxidative damage in a dose dependent manner. Further, geraniin induced the expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase-1 (NQO1) and level of glutathione (GSH) in a concentration- and time-dependent manner, and increased Nrf2 nuclear translocation. The Nrf2-related cytoprotective effects of geraniin were PI3K/AKT and extracellular signal-regulated protein kinase1/2 (ERK1/2) pathway-dependent. However, inhibitors of PI3K/AKT and ERK1/2 (LY294002 or U0126) not only suppressed geraniin-induced nuclear translocation of Nrf2 but also abolished the expression of HO-1, NQO1 and GSH.These results demonstrated that geraniin induced Nrf2-mediated expression of antioxidant enzymes HO-1 and NQO1, presumably via PI3K/AKT and ERK1/2 signaling pathways, thereby protecting cells from H2O2-induced oxidative cell death.Geraniin, at least in part, offers an antioxidant defense capacity to protect cells from the oxidative stress-related diseases.Display Omitted
Keywords: Nrf2; Human hepatocarcinoma cell line; Antioxidant gene; Geraniin; Oxidative stress; Cytoprotection;

Lichen endophyte derived pyridoxatin inactivates Candida growth by interfering with ergosterol biosynthesis by Wenqiang Chang; Ming Zhang; Ying Li; Xiaobin Li; Yanhui Gao; Zhiyu Xie; Hongxiang Lou (1762-1771).
This study is to characterize the antifungal effects of pyridoxatin (PYR), a small natural product isolated from an endolichenic fungus.The susceptibility tests in vitro and in vivo by using Caenorhabditis elegans as an infectious model were performed to evaluate the antifungal efficacy of PYR against Candida species. The cytotoxicity of PYR against normal human cells was tested using MTT assay. The transcriptional levels of genes related to sterol synthesis and cell cycle regulation were measured using real-time quantitative PCR (qPCR). The contents ergosterol, squalene, lanosterol were detected by liquid chromatography/tandem mass spectrometry (LC/MS).PYR was effective against four tested Candida species with its minimal inhibitory concentrations (MICs) ranging from 1–4 μg/ml. No obvious cytotoxicity was observed for PYR against normal human cells. PYR inhibited the growth of Candida albicans, preventing the biofilm formation. And the antifungal action was independent on efflux pumps. The in vivo test showed PYR greatly prolonged the survival of infected C. elegans. qPCR results revealed that most of the genes related to sterol biosynthesis were considerably down-regulated in PYR-treated cells. Determination of the sterol content found that PYR inhibited the ergosterol synthesis dose dependently and caused the accumulation of squalene and lanosterol. Moreover, analysis of the structure–activity relationship revealed the heterocyclic hydroxamic acid in PYR was the key group for the antifungal action.PYR interferes with the ergosterol synthesis to exert antifungal action.The elucidated mechanism provides possible applications of PYR in fighting clinical relevant fungal infections.
Keywords: Candida; Ergosterol; Pyridoxatin; Heterocyclic hydroxamic acid;

Tryptophan catabolism along the kynurenine pathway is associated with a number of pathologies including cataract formation and cancer. Whilst the chemical reactions of kynurenine are well studied, less is known about the reactivity of its precursor N-formylkynurenine (NFK). We previously reported the generation of a strong fluorophore in an aqueous reaction of NFK with piperidine, and herein we describe its structure and mechanism of formation.Compounds were identified using NMR, mass and UV spectroscopic techniques. The products from the reaction of amines with amino acids were quantified using HPLC-MS.The novel fluorophore was identified as a tetrahydroquinolone adduct (PIP-THQ), where piperidine is N-formylated and attached at its 2-position to the quinolone. NFK is initially deaminated to generate an unsaturated enone, which forms an adduct with piperidine and is subsequently converted into the fluorophore. Testing of a variety of other secondary amines showed that only cyclic amines unsubstituted at both positions adjacent to nitrogen could form fluorophores efficiently. The amino acids tryptophan and kynurenine, which lack the formamide group do not form such fluorophores.NFK forms fluorophores in a not previously published reaction with cyclic amines.Our study is the first to provide evidence for concurrent transamidation and substitution at the 2-position of a cyclic amine occurring under moderately-heated aqueous conditions with no added catalysts. The high reactivity of NFK demonstrated here could result in formation of biologically relevant metabolites yet to be characterised.Display Omitted
Keywords: Indoleamine 2,3-dioxygenase; N-formylkynurenine; Tryptophan catabolism; Transamidation; Piperidine; Fluorophore;

Since the initial discovery of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in a large subset of human low-grade gliomas and acute myelogenous leukemia (AML), much interest focused on the function of IDH1 and on the relationship between mutations in IDH1 and tumor progression. To date, mutations in the IDH1 gene have been found in numerous cancers with the highest frequencies occurring in gliomas, chondrosarcomas/enchondromas and cholangiocarcinomas.IDH1 was first described in the scientific literature as early as 1950. Early researchers proposed that the enzyme likely functions in cellular lipid metabolism based on the observation that the enzymatic reaction produces NADPH and partially localizes to peroxisomes. This article highlights the studies implicating IDH1 in cytoplasmic and peroxisomal lipid metabolism from the early researchers to the recent studies examining mutant IDH1R132, the most common IDH1 mutation found in cancer.While a role for IDH1 in lipid biosynthesis in the liver and adipose tissue is now established, a role in lipid metabolism in the brain and tumors is beginning to be examined. The recent discoveries that IDH1R132H interferes with the metabolism of phospholipids in gliomas and that IDH1 activity could participate in the synthesis of acetyl-CoA from glutamine in hypoxic tumors highlight roles for IDH1 in lipid metabolism in a broad spectrum of tissues.Interferences in cytoplasmic and peroxisomal lipid metabolism by IDH1R132 may contribute to the more favorable clinical outcome in patients whose tumors express mutations in the IDH1 gene.
Keywords: IDH1R132; Lipid; Peroxisome; Plasmalogen; Cancer;

Aquaporins are responsible for water transport across lipid membranes. They are also able to transport reactive oxygen species, playing an important role in redox signaling. Certain plant aquaporins have even the ability to be regulated by oxidative stress. However, the underlying mechanisms are still not fully understood.Here, molecular dynamics simulations were employed to determine the activation free energies related to the transport of reactive oxygen species through both mammalian and plant aquaporin models.Both aquaporins may transport hydrogen peroxide (H2O2) and the protonated form of superoxide radicals (HO2). The solution-to-pore transfer free energies were low for small oxy-radicals, suggesting that even highly reactive hydroxyl radicals (HO) might have access to the pore interior and oxidize amino acids responsible for channel selectivity. In the plant aquaporin, no significant change in water permeability was observed upon oxidation of the solvent-exposed disulfide bonds at the extracellular region. During the simulated time scale, the existence of a direct oxidative gating mechanism involving these disulfide bonds could not be demonstrated.Simulation results may improve the understanding of redox signaling mechanisms and help in the interpretation of protein oxidative labeling experiments.Display Omitted
Keywords: Aquaporin; Biomembrane; Molecular dynamics simulation; Oxidative labeling; Reactive oxygen species; Redox signaling;

Mn/Fe-superoxide dismutase (SOD) is a family of enzymes essential for organisms to be able to cope with oxygen. These enzymes bound to their classical metals catalyze the dismutation of the free radical superoxide anion (O2) to H2O2 and molecular oxygen. E. coli has the manganese-dependent SOD A and the iron-dependent SOD B.Strains of E. coli overexpressing SOD A or SOD B were grown in media with different metal compositions. SODs were purified and their metal content and SOD activity were determined. Those proteins were incubated with H2O2 and assayed for oxidation of Amplex red or o-phenylenediamine, consumption of H2O2, release of iron and protein radical formation. Cell survival was determined in bacteria with MnSOD A or FeSOD A after being challenged with H2O2.We show for the first time that the bacterial manganese-dependent SOD A when bound to iron (FeSOD A) has peroxidase activity. The in vivo formation of the peroxidase FeSOD A was increased when media had higher levels of iron because of a decreased manganese metal incorporation. In comparison to bacteria with MnSOD A, cells with FeSOD A had a higher loss of viability when exposed to H2O2.The biological occurrence of this fundamental antioxidant enzyme in an alternative iron-dependent state represents an important source of free radical formation.Display Omitted
Keywords: Superoxide dismutase (SOD); Peroxidase; Free radical; Metal; Iron; Manganese;

Identification and mechanism of action analysis of the new PARP-1 inhibitor 2″-hydroxygenkwanol A by Fabrizio Dal Piaz; Piera Ferro; Antonio Vassallo; Michele Vasaturo; Giovanni Forte; Maria Giovanna Chini; Giuseppe Bifulco; Alessandra Tosco; Nunziatina De Tommasi (1806-1814).
Poly(ADP-ribose) polymerase 1 (PARP-1) activity has been implicated in the pathogenesis of numerous diseases as cancer, inflammation, diabetes and neurodegenerative disorders, therefore the research for new PARP-1 inhibitors is still an active area.To identify new potential PARP-1 inhibitors, we performed a screening of a small-molecule library consisting of polyphenols isolated from plants used in the traditional medicine, by Surface Plasmon Resonance (SPR). Biochemical and cellular assays were performed to confirm SPR results and select the promising candidate(s). Finally, limited proteolysis and ligand docking analyses allowed defining the protein region involved in the interaction with the putative inhibitor(s).The dimeric spiro-flavonoid 2″-hydroxygenkwanol A, member of a relatively recently discovered class of flavonoids containing a spirane C-atom, has been identified as possible PARP-1 inhibitor. This compound showed a high affinity for the polymerase (KD: 0.32 ± 0.05 μM); moreover PARP-1 activity in the presence of 2″-hydroxygenkwanol A was significantly affected both when using the recombinant protein and when measuring the cellular effects. Finally, our study suggests this compound to efficiently interact with the protein catalytic domain, into the nicotine binding pocket.2″-hydroxygenkwanol A efficiently binds and inhibits PARP-1 at submicromolar concentrations, thus representing a promising lead for the design of a new class of PARP-1 modulators, useful as therapeutic agents and/or biochemical tools.Our study has identified an additional class of plant molecules, the spiro-biflavonoids, with known beneficial pharmacological properties but with an unknown mechanism of action, as a possible novel class of PARP-1 activity inhibitors.
Keywords: PARP-1; Biflavonoids; Plant compounds; Surface Plasmon Resonance; Mass spectrometry; Molecular modeling;

Maackia amurensis leukoagglutinin (MAL) is a glycoprotein and sialic acid-binding lectin that is used widely in the detection and characterization of sialoglycoconjugates and human cancer cells. However, its N-linked glycan structure and role have yet to be determined.The N-linked glycans were analyzed using high-performance liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the secondary structure was investigated using circular dichroism analysis. A hemagglutination assay was performed. Furthermore, surface plasmon resonance analysis, and fluorescence microscopy and fluorescence-activated cell-sorting analysis were conducted to assess the sialoglycoprotein-binding ability and its usefulness in the detection of human breast cancer MCF-7 cells, respectively.Analysis of the N-linked glycan structure of MAL confirmed the presence of eight glycans, comprising two α1,3-fucosylated paucimannosidic-type and six high-mannose-type glycans. Glycan analysis of MAL that had been treated with peptide N-glycosidase F (de-M-MAL) revealed that while the two α1,3-fucosylated paucimannosidic glycans remained attached following the treatment, the six high-mannose-type glycans had been completely cleaved from the original MAL. There were almost no secondary structural changes between MAL and de-M-MAL; however, the lectin activities exhibited by MAL, such as hemagglutination and binding to a sialoglycoprotein, were completely absent in de-M-MAL, and the ability to detect human breast cancer MCF-7 cells was 77% lower in de-M-MAL than in MAL.The high-mannose-type glycans in intact MAL are closely associated with its lectin activities.This is the first report of the N-linked glycan structure of MAL and the effect of high-mannose-type glycans on lectin activities.
Keywords: Maackia amurensis lectin; N-linked glycan; High-mannose-type; Activity;

Macromolecular crowding: Macromolecules friend or foe by Shruti Mittal; Rimpy Kaur Chowhan; Laishram Rajendrakumar Singh (1822-1831).
Cellular interior is known to be densely crowded due to the presence of soluble and insoluble macromolecules, which altogether occupy ~ 40% of the total cellular volume. This results in altered biological properties of macromolecules.Macromolecular crowding is observed to have both positive and negative effects on protein folding, structure, stability and function. Significant data has been accumulated so far on both the aspects. However, most of the review articles so far have focused on the positive aspect of macromolecular crowding and not much attention has been paid on the deleterious aspect of crowding on macromolecules. In order to have a complete knowledge of the effect of macromolecular crowding on proteins and enzymes, it is important to look into both the aspects of crowding to determine its precise role under physiological conditions. To fill the gap in the understanding of the effect of macromolecular crowding on proteins and enzymes, this review article focuses on the deleterious influence of crowding on macromolecules.Macromolecular crowding is not always good but also has several deleterious effects on various macromolecular properties. Taken together, the properties of biological macromolecules in vivo appears to be finely regulated by the nature and level of the intracellular crowdedness in order to perform their biological functions appropriately.The information provided here gives an understanding of the role played by the nature and level of cellular crowdedness in intensifying and/or alleviating the burden of various proteopathies.
Keywords: Protein folding; Protein aggregation; Enzyme activity; Protein stability; Excluded volume effect;

Actin-cytoskeleton polymerization differentially controls the stability of Ski and SnoN co-repressors in normal but not in transformed hepatocytes by Cassandre Caligaris; Genaro Vázquez-Victorio; Marcela Sosa-Garrocho; Diana G. Ríos-López; Alvaro Marín-Hernández; Marina Macías-Silva (1832-1841).
Ski and SnoN proteins function as transcriptional co-repressors in the TGF-β pathway. They regulate cell proliferation and differentiation, and their aberrant expression results in altered TGF-β signalling, malignant transformation, and alterations in cell proliferation.We carried out a comparative characterization of the endogenous Ski and SnoN protein regulation by TGF-β, cell adhesion disruption and actin-cytoskeleton rearrangements between normal and transformed hepatocytes; we also analyzed Ski and SnoN protein stability, subcellular localization, and how their protein levels impact the TGF-β/Smad-driven gene transcription.Ski and SnoN protein levels are lower in normal hepatocytes than in hepatoma cells. They exhibit a very short half-life and a nuclear/cytoplasmic distribution in normal hepatocytes opposed to a high stability and restricted nuclear localization in hepatoma cells. Interestingly, while normal cells exhibit a transient TGF-β-induced gene expression, the hepatoma cells are characterized by a strong and sustained TGF-β-induced gene expression. A novel finding is that Ski and SnoN stability is differentially regulated by cell adhesion and cytoskeleton rearrangements in the normal hepatocytes. The inhibition of protein turnover down-regulated both Ski and SnoN co-repressors impacting the kinetic of expression of TGF-β-target genes.Normal regulatory mechanisms controlling Ski and SnoN stability, subcellular localization and expression are altered in hepatocarcinoma cells.This work provides evidence that Ski and SnoN protein regulation is far more complex in normal than in transformed cells, since many of the normal regulatory mechanisms are lost in transformed cells.Display Omitted
Keywords: TGF-β; Ski; SnoN; Protein stability; Transcriptional regulation; Hepatocarcinoma;

Kinetic characterization of human histone H3 lysine 36 methyltransferases, ASH1L and SETD2 by Mohammad S. Eram; Ekaterina Kuznetsova; Fengling Li; Evelyne Lima-Fernandes; Steven Kennedy; Irene Chau; Cheryl H. Arrowsmith; Matthieu Schapira; Masoud Vedadi (1842-1848).
Dysregulation of methylation of lysine 36 on histone H3 (H3K36) have been implicated in a variety of diseases including cancers. ASH1L and SETD2 are two enzymes among others that catalyze H3K36 methylation. H3K4 methylation has also been reported for ASH1L.Radioactivity-based enzyme assays, Western and immunoblotting using specific antibodies and molecular modeling were used to characterize substrate specificity of ASH1L and SETD2.Here we report on the assay development and kinetic characterization of ASH1L and SETD2 and their substrate specificities in vitro. Both enzymes were active with recombinant nucleosome as substrate. However, SETD2 but not ASH1L methylated histone peptides as well indicating that the interaction of the basic post-SET extension with substrate may not be critical for SETD2 activity. Both enzymes were not active with nucleosome containing a H3K36A mutation indicating their specificity for H3K36. Analyzing the methylation state of the products of ASH1L and SETD2 reactions also confirmed that both enzymes mono- and dimethylate H3K36 and are inactive with H3K4 as substrate, and that only SETD2 is able to trimethylate H3K36 in vitro.We determined the kinetic parameters for ASH1L and SETD2 activity enabling screening for inhibitors that can be used to further investigate the roles of these two proteins in health and disease. Both ASH1L and SETD2 are H3K36 specific methyltransferases but only SETD2 can trimethylate this mark. The basic post-SET extension is critical for ASH1L but not SETD2 activity.We provide full kinetic characterization of ASH1L and SETD2 activity.
Keywords: ASH1L; SETD2; Assay development; Screening; H3K36;

Kahalalide F (KF) and its isomer iso-kahalalide F (isoKF), both of which can be isolated from the mollusk Elysia rufescens and its diet alga Bryopsis pennata, are potent cytotoxic agents that have advanced through five clinical trials. Due to a short half-life, narrow spectrum of activity, and a modest response in patients, further efforts to modify the molecule are required to address its limitations. In addition, due to the high cost in producing KF analogues using solid phase peptide synthesis (SPPS), a degradation and reconstruction approach was employed using natural KF from a seasonal algal bloom to generate KF analogues. N-protected KF was carefully hydrolyzed at the amide linkage between l-Thr12 and d-Val13 using dilute HCl. The synthesis of the C-terminal fragment began with the formation of hexanoic succinimide ester, followed by a reaction with dipeptides. The final coupling reaction was performed between the semisynthesized Fmoc–KF hydrolysis product and the C-terminal fragment, followed by the deprotection of the Fmoc group.Six KF analogues with an addition of an amino acid residue on the N-terminal chain, d-Val14–isoKF (2), Val13–Val14–isoKF (3), d-Leu14–isoKF (4), d-Pro14–isoKF (5), d-Phe14–isoKF (6), and 3,4-2F-d-Phe14-isoKF (7) were prepared using semisynthesis at the exposed N-terminal chain.The overall yield of the medication was 45%. This approach is economical, efficient and amendable to large-scale production while eliminated a nuisance algal bloom. B. pennata blooms are capable of producing KF in good yields. The semisynthesis from the natural product produced N-terminal modifications for the construction of inexpensive semisynthetic KF libraries.Display Omitted
Keywords: Kahalalide F; Partial hydrolysis; N-terminal modification;

FASCIN and alpha-actinin can regulate the conformation of actin filaments by Katalin Türmer; József Orbán; Pál Gróf; Miklós Nyitrai (1855-1861).
Actin filament bundling proteins mediate numerous processes in cells such as the formation of cell membrane protrusions or cell adhesions and stress fiber based locomotion. Among them alpha-actinin and fascin are the most abundant ones. This work characterizes differences in molecular motions in actin filaments due to the binding of these two actin bundling proteins.We investigated how alpha-actinin and fascin binding modify the conformation of actin filaments by using conventional and saturation transfer EPR methods.The result characteristic for motions on the microsecond time scale showed that both actin bundling proteins made the bending and torsional twisting of the actin filaments slower. When nanosecond time scale molecular motions were described the two proteins were found to induce opposite changes in the actin filaments. The binding of one molecule of alpha-actinin or fascin modified the conformation of numerous actin protomers.As fascin and alpha-actinin participates in different cellular processes their binding can serve the proper tuning of the structure of actin by establishing the right conformation for the interactions with other actin binding proteins. Our observations are in correlation with the model where actin filaments fulfill their biological functions under the regulation by actin-binding proteins.Supporting the general model for the cellular regulation of the actin cytoskeleton we showed that two abundant actin bundling proteins, fascin and alpha-actinin, alter the conformation of actin filaments through long range allosteric interactions in two different ways providing the structural framework for the adaptation to specific biological functions.
Keywords: Actin; Bundle; Fascin; Alpha-actinin; EPR spectroscopy;

Single-cell analysis of mast cell degranulation induced by airway smooth muscle-secreted chemokines by Benjamin M. Manning; Audrey F. Meyer; Sarah M. Gruba; Christy L. Haynes (1862-1868).
Asthma is a chronic inflammatory disease characterized by narrowed airways, bronchial hyper-responsiveness, mucus hyper-secretion, and airway remodeling. Mast cell (MC) infiltration into airway smooth muscle (ASM) is a defining feature of asthma, and ASM regulates the inflammatory response by secreting chemokines, including CXCL10 and CCL5. Single cell analysis offers a unique approach to study specific cellular signaling interactions within large and complex signaling networks such as the inflammatory microenvironment in asthma.Carbon-fiber microelectrode amperometry was used to study the effects of ASM-secreted chemokines on mouse peritoneal MC degranulation.MC degranulation in response to CXCL10 and CCL5 was monitored at the single cell level. Relative to IgE-mediated degranulation, CXCL10- and CCL5-stimulated MCs released a decreased amount of serotonin per granule with fewer release events per cell. Decreased serotonin release per granule was correlated with increased spike half-width and rise-time values.MCs are directly activated by ASM-associated chemokines. CXCL10 and CCL5 induce less robust MC degranulation compared to IgE- and A23187-stimulation. The kinetics of MC degranulation are signaling pathway-dependent, suggesting a biophysical mechanism of regulated degranulation that incorporates control over granule trafficking, transport, and docking machinery.The biophysical mechanisms, including variations in number of exocytotic release events, serotonin released per granule, and the membrane kinetics of exocytosis that underlie MC degranulation in response to CXCL10 and CCL5 were characterized at the single cell level. These findings clarify the function of ASM-derived chemokines as instigators of MC degranulation relative to classical mechanisms of MC stimulation.
Keywords: Carbon-fiber microelectrode amperometry; Exocytosis; Asthma;

The discovery of stable, yet functional, protein mutants is a limiting factor in the development of biotechnological applications, structural studies or in drug discovery. Rapid detection of functional mutants is especially challenging for water channel aquaporins, as they do not have a directly measurable enzymatic or binding activity. Current methods available are time consuming and only applicable to specific aquaporins.Herein we describe an assay based on the protective effect of aquaporins on yeast S. cerevisiae in response to rapid freezing.Yeast overexpressing a functional water-permeable aquaporin of choice are rescued after the challenge, while inactive or blocked aquaporins confer no protection and lead to cell death. The potential of this assay is shown by screening a small number of E. coli aquaporin Z (AQPZ) mutants. Additionally, a library of ~ 10,000 drug-like compounds was tested against human AQP1 (hAQP1).Since rescue is only dependent on transmembrane water flux, the assay is applicable to water-permeable aquaporins of any origin.Mapping of permissive mutations on the aquaporin structure can help delineate the minimal requirements for effective water transport. Alternatively, the assay can be potentially used to discover compounds that inhibit aquaporin water transport. When additionally screened for thermostability, functional aquaporin mutants can be useful in the development of biomimetic membranes for water purification, or to improve the likelihood of producing well-diffracting crystals, enabling rational design of much needed aquaporin inhibitors.Display Omitted
Keywords: Yeast; Human aquaporin 1; Aquaporin Z; Freeze–thawing; Drug discovery; Functional mutant selection;

The genome of poplar (Populus trichocarpa) encodes 81 glutathione transferases (GSTs) annotated in eight distinct classes. The tau class is considered the most versatile in the biotransformation of xenobiotics and is composed of 58 GSTs. Two of the enzymes, GSTU16 and GSTU45, have particular interest since their expression is induced by exposure of poplar tissues to 2,4,6-trinitrotoluene (TNT) and could potentially be involved in the metabolism of this toxic environmental contaminant.DNA encoding these GSTs was synthesized and the proteins were heterologously expressed in Escherichia coli and the purified enzymes were characterized.GSTU16 assayed with a number of conventional GST substrates showed the highest specific activity (60 μmol min− 1  mg− 1) with phenethyl isothiocyanate, 150-fold higher than that with CDNB. By contrast, GSTU45 showed CDNB as the most active substrate (3.3 μmol min− 1  mg− 1) whereas all of the 16 alternative substrates tested yielded significantly lower activities. Homology modeling suggested that the aromatic residues Phe10 and Tyr107 in the active site of GSTU16 are promoting the high activity with PEITC and other substrates with aromatic side-chains. Nonetheless, TNT was a poor substrate for GSTU16 as well as for GSTU45 with a specific activity of 0.05 nmol min− 1  mg− 1 for both enzymes.GSTU16 and GSTU45 do not play a major role in the degradation of TNT in poplar.
Keywords: Glutathione transferase; Detoxication; Populus trichocarpa; Phytoremediation; TNT;

Anle138b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to α-synuclein aggregates by Andreas A. Deeg; Anne M. Reiner; Felix Schmidt; Florian Schueder; Sergey Ryazanov; Viktoria C. Ruf; Karin Giller; Stefan Becker; Andrei Leonov; Christian Griesinger; Armin Giese; Wolfgang Zinth (1884-1890).
Special diphenyl-pyrazole compounds and in particular anle138b were found to reduce the progression of prion and Parkinson's disease in animal models. The therapeutic impact of these compounds was attributed to the modulation of α-synuclein and prion-protein aggregation related to these diseases.Photophysical and photochemical properties of the diphenyl-pyrazole compounds anle138b, anle186b and sery313b and their interaction with monomeric and aggregated α-synuclein were studied by fluorescence techniques.The fluorescence emission of diphenyl-pyrazole is strongly increased upon incubation with α-synuclein fibrils, while no change in fluorescence emission is found when brought in contact with monomeric α-synuclein. This points to a distinct interaction between diphenyl-pyrazole and the fibrillar structure with a high binding affinity (Kd  = 190 ± 120 nM) for anle138b. Several α-synuclein proteins form a hydrophobic binding pocket for the diphenyl-pyrazole compound. A UV-induced dehalogenation reaction was observed for anle138b which is modulated by the hydrophobic environment of the fibrils.Fluorescence of the investigated diphenyl-pyrazole compounds strongly increases upon binding to fibrillar α-synuclein structures. Binding at high affinity occurs to hydrophobic pockets in the fibrils.The observed particular fluorescence properties of the diphenyl-pyrazole molecules open new possibilities for the investigation of the mode of action of these compounds in neurodegenerative diseases. The high binding affinity to aggregates and the strong increase in fluorescence upon binding make the compounds promising fluorescence markers for the analysis of aggregation-dependent epitopes.
Keywords: Protein aggregation; Amyloid dye; Anle138b; α-Synuclein; Fluorescence marker; Parkinson's disease;

Leishmania infantum trypanothione reductase is a promiscuous enzyme carrying an NADPH:O2 oxidoreductase activity shared by glutathione reductase by Gabriella Angiulli; Antonella Lantella; Elena Forte; Francesco Angelucci; Gianni Colotti; Andrea Ilari; Francesco Malatesta (1891-1897).
Leishmania infantum is a protozoan of the trypanosomatid family causing visceral leishmaniasis. Leishmania parasites are transmitted by the bite of phlebotomine sand flies to the human host and are phagocyted by macrophages. The parasites synthesize N1-N8-bis(glutationyl)-spermidine (trypanothione, TS2), which furnishes electrons to the tryparedoxin-tryparedoxin peroxidase couple to reduce the reactive oxygen species produced by macrophages. Trypanothione is kept reduced by trypanothione reductase (TR), a FAD-containing enzyme essential for parasite survival.The enzymatic activity has been studied by stopped-flow, absorption spectroscopy, and amperometric measurements.The study reported here demonstrates that the steady-state parameters change as a function of the order of substrates addition to the TR-containing solution. In particular, when the reaction is carried out by adding NADPH to a solution containing the enzyme and trypanothione, the KM for NADPH decreases six times compared to the value obtained by adding TS2 as last reagent to start the reaction (1.9 vs. 12 μM). More importantly, we demonstrate that TR is able to catalyze the oxidation of NADPH also in the absence of trypanothione. Thus, TR catalyzes the reduction of O2 to water through the sequential formation of C(4a)-(hydro)peroxyflavin and sulfenic acid intermediates. This NADPH:O2 oxidoreductase activity is shared by Saccharomyces cerevisiae glutathione reductase (GR).TR and GR, in the absence of their physiological substrates, may catalyze the electron transfer reaction from NADPH to molecular oxygen to yield water.TR and GR are promiscuous enzymes.Display Omitted
Keywords: Trypanothione reductase; Sulfenic acid; Electron transfer; O2 reduction; Glutathione reductase; Promiscuous enzymes;

Role of receptor for advanced glycation end products in the complication and progression of various types of cancers by Parth Malik; Narender Chaudhry; Rashmi Mittal; Tapan K. Mukherjee (1898-1904).
Receptor for advanced glycation end-products popularly known as RAGE is a cell surface immunoglobulin class of molecule, binds with multiple ligands and therefore considered as a multi-ligand receptor. Use of RAGE deficient mice (RAGE−/−) as well as established mouse models pertaining to inflammation-associated carcinogenesis such as that of chemically induced carcinogenesis and colitis associated cancer provides a direct genetic evidence for a likelihood novel role of RAGE in cancer, with respect to its ability to lead cancer cell proliferation and survival. Besides inflammation, interaction of RAGE with its various ligands enhances oxidative stress both in cancerous and noncancerous cells which further complicates the progression of cancers.Till date, no single review article has discussed the mechanism of RAGE dependent complication of cancers, particularly the role of RAGE in cancer cell proliferation, angiogenesis, survival and anti-apoptosis needs to be discussed.RAGE enhances the number of cancer cells by activating the cell cycle proteins (e.g., cyclin D1), anti-apoptotic proteins (e.g., BCl2), prosurvival (AKT) and autophagic proteins. Role of RAGE has also been detected in formation of new blood vessels (angiogenesis) in the cancer cells and activation of myeloid derived suppressor cells (MDSCs).This review article describes the role of RAGE in the complication of various types of cancers and the possible usefulness of RAGE dependent therapy to confront cancers in a stronger magnitude.Display Omitted
Keywords: RAGE; Cancer; Ligands; Inflammation; Cancer therapy;

Intracellular pH imaging in cancer cells in vitro and tumors in vivo using the new genetically encoded sensor SypHer2 by Marina V. Shirmanova; Irina N. Druzhkova; Maria M. Lukina; Mikhail E. Matlashov; Vsevolod V. Belousov; Ludmila B. Snopova; Natalia N. Prodanetz; Varvara V. Dudenkova; Sergey A. Lukyanov; Elena V. Zagaynova (1905-1911).
Measuring intracellular pH (pHi) in tumors is essential for the monitoring of cancer progression and the response of cancer cells to various treatments. The purpose of the study was to develop a method for pHi mapping in living cancer cells in vitro and in tumors in vivo, using the novel genetically encoded indicator, SypHer2.A HeLa Kyoto cell line stably expressing SypHer2 in the cytoplasm was used, to perform ratiometric (dual excitation) imaging of the probe in cell culture, in 3D tumor spheroids and in tumor xenografts in living mice.Using SypHer2, pHi was demonstrated to be 7.34 ± 0.11 in monolayer HeLa cells in vitro under standard cultivation conditions. An increasing pHi gradient from the center to the periphery of the spheroids was displayed. We obtained fluorescence ratio maps for HeLa tumors in vivo and ex vivo. Comparison of the map with the pathomorphology and with hypoxia staining of the tumors revealed a correspondence of the zones with higher pHi to the necrotic and hypoxic areas.Our results demonstrate that pHi imaging with the genetically encoded pHi indicator, SypHer2, can be a valuable tool for evaluating tumor progression in xenograft models.We have demonstrated, for the first time, the possibility of using the genetically encoded sensor SypHer2 for ratiometric pH imaging in cancer cells in vitro and in tumors in vivo. SypHer2 shows great promise as an instrument for pHi monitoring able to provide high accuracy and spatiotemporal resolution.
Keywords: Intracellular pH; Genetically encoded sensor; SypHer2; Cancer cell; Tumor; Ratiometric imaging;

AKAP3 degradation in sperm capacitation is regulated by its tyrosine phosphorylation by Ruth Vizel; Pnina Hillman; Debby Ickowicz; Haim Breitbart (1912-1920).
The A-kinase anchoring protein (AKAP) family is essential for sperm motility, capacitation and the acrosome reaction. PKA-dependent protein tyrosine phosphorylation occurs in mammalian sperm capacitation including AKAP3. In a recent study, we showed that AKAP3 undergoes degradation under capacitation conditions. Thus, we tested here whether AKAP3 degradation might be regulated by its tyrosine phosphorylation.The intracellular levels of AKAP3 were determined by western blot (WB) analysis using specific anti-AKAP3 antibodies. Tyrosine phosphorylation of AKAP3 was tested by immunoprecipitation and WB analysis. Acrosome reaction was examined using FITC-pisum sativum agglutinin.AKAP3 is degraded and undergoes tyrosine-dephosphorylation during sperm capacitation and the degradation was reduced by inhibition of tyrosine phosphatase and enhanced by inhibition of tyrosine kinase. Sperm starvation or inhibition of mitochondrial respiration, which reduce cellular ATP levels, significantly accelerated AKAP3 degradation. Treatment with vanadate, or Na+ or bicarbonate depletion, reduced AKAP3-degradation and the AR rate, while antimycin A or NH4Cl elevated both AKAP3-degradation and the AR degree. Treatment of sperm with NH4Cl enhanced PKA-dependent phosphorylation of four proteins, further supporting the involvement of AKAP3-degradation in capacitation. To demonstrate more specifically that sperm capacitation requires AKAP3-degradation, we inhibited AKAP3-degradation using anti-AKAP3 antibody in permeabilized cells. The anti-AKAP3-antibody induced significant inhibition of AKAP3-degradation and of the AR rate.Sperm capacitation process requires AKAP3-degradation, and the degradation degree is regulated by the level of AKAP3 tyrosine phosphorylation.Better understanding of the molecular mechanisms that mediate sperm capacitation can be used for infertility diagnosis, treatment and the developing of male contraceptives.
Keywords: Sperm; Capacitation; Acrosome reaction; AKAP3; Degradation;

High yield expression and purification of equilibrative nucleoside transporter 7 (ENT7) from Arabidopsis thaliana by Christopher Girke; Elena Arutyunova; Maria Syed; Michaela Traub; Torsten Möhlmann; M. Joanne Lemieux (1921-1929).
Equilibrative nucleoside transporters (ENTs) facilitate the import of nucleosides and their analogs into cells in a bidirectional, non-concentrative manner. However, in contrast to their name, most characterized plant ENTs act in a concentrative manner. A direct characterization of any ENT protein has been hindered due to difficulties in overexpression and obtaining pure recombinant protein.The equilibrative nucleoside transporter 7 from Arabidopsis thaliana (AtENT7) was expressed in Xenopus laevis oocytes to assess mechanism of substrate uptake. Recombinant protein fused to enhanced green fluorescent protein (eGFP) was expressed in Pichia pastoris to characterize its oligomeric state by gel filtration and substrate binding by microscale thermophoresis (MST).AtENT7 expressed in X. laevis oocytes works as a classic equilibrative transporter. The expression of AtENT7-eGFP in the P. pastoris system yielded milligram amounts of pure protein that exists as stable homodimers. The concentration dependent binding of purine and pyrimidine nucleosides to the purified recombinant protein, assessed by MST, confirmed that AtENT7-eGFP is properly folded. For the first time the binding of nucleobases was observed for AtENT7.The availability of pure recombinant AtENT7 will permit detailed kinetic and structural studies of this unique member of the ENT family and, given the functional similarity to mammalian ENTs, will serve as a good model for understanding the structural basis of translocation mechanism for the family.
Keywords: Equilibrative nucleoside transporter; Arabidopsis thaliana; ENT7; Nucleoside; Nucleobase; Membrane transport; Recombinant expression; Reconstitution; Microscale thermophoresis; MST; Fluorescence size exclusion chromatography; FSEC;

Heme interplay between IlsA and IsdC: Two structurally different surface proteins from Bacillus cereus by Elise Abi-Khalil; Diego Segond; Tyson Terpstra; Gwenaëlle André-Leroux; Mireille Kallassy; Didier Lereclus; Fadi Bou-Abdallah; Christina Nielsen-Leroux (1930-1941).
Iron is an essential element for bacterial growth and virulence. Because of its limited bioavailability in the host, bacteria have adapted several strategies to acquire iron during infection. In the human opportunistic bacteria Bacillus cereus, a surface protein IlsA is shown to be involved in iron acquisition from both ferritin and hemoproteins. IlsA has a modular structure consisting of a NEAT (Near Iron transporter) domain at the N-terminus, several LRR (Leucine Rich Repeat) motifs and a SLH (Surface Layer Homology) domain likely involved in anchoring the protein to the cell surface.Isothermal titration calorimetry, UV–Vis spectrophotometry, affinity chromatography and rapid kinetics stopped-flow measurements were employed to probe the binding and transfer of hemin between two different B. cereus surface proteins (IlsA and IsdC).IlsA binds hemin via the NEAT domain and is able to extract heme from hemoglobin whereas the LRR domain alone is not involved in these processes. A rapid hemin transfer from hemin-containing IlsA (holo-IlsA) to hemin-free IsdC (apo-IsdC) is demonstrated.For the first time, it is shown that two different B. cereus surface proteins (IlsA and IsdC) can interact and transfer heme suggesting their involvement in B. cereus heme acquisition.An important role for the complete Isd system in heme-associated bacterial growth is demonstrated and new insights into the interplay between an Isd NEAT surface protein and an IlsA-NEAT-LRR protein, both of which appear to be involved in heme-iron acquisition in B. cereus are revealed.
Keywords: Hemin binding; IlsA NEAT domain; Hemoglobin; Kinetics; LRR domain;

Protein–RNA and protein–glycan recognitions in light of amino acid codes by Jozef Nahalka; Eva Hrabarova; Klaudia Talafova (1942-1952).
RNA-binding proteins, in cooperation with non-coding RNAs, play important roles in post-transcriptional regulation. Non-coding micro-RNAs control information flow from the genome to the glycome by interacting with glycan-synthesis enzymes. Glycan-binding proteins read the cell surface and cytoplasmic glycome and transfer signals back to the nucleus. The profiling of the protein–RNA and protein–glycan interactomes is of significant medicinal importance.This review discusses the state-of-the-art research in the protein–RNA and protein–glycan recognition fields and proposes the application of amino acid codes in profiling and programming the interactomes.The deciphered PUF–RNA and PPR–RNA amino acid recognition codes can be explained by the protein–RNA amino acid recognition hypothesis based on the genetic code. The tripartite amino acid code is also involved in protein–glycan interactions. At present, the results indicate that a system of four codons (“gnc”, where n = g — guanine, c — cytosine, u — uracil or a — adenine) and four amino acids (G — glycine, A — alanine, V — valine, D — aspartic acid) could be the original genetic code that imprinted “rules” into both recognition processes.Amino acid recognition codes have provocative potential in the profiling and programming of the protein–RNA and protein–glycan interactomes. The profiling and even programming of the interactomes will play significant roles in diagnostics and the development of therapeutic procedures against cancer and neurodegenerative, developmental and other diseases.
Keywords: Protein–RNA interactome; Protein–glycan interactome; Amino acid codes; Computational profiling; Protein engineering; Cell developmental diseases;

Structural and biochemical insights into the degradation mechanism of chitosan by chitosanase OU01 by Qianqian Lyu; Yanhong Shi; Song Wang; Yan Yang; Baoqin Han; Wanshun Liu; David N.M. Jones; Weizhi Liu (1953-1961).
A detailed knowledge about the degradation mechanism of chitosanase hydrolysis is critical for the design of novel enzymes to produce well-defined chito-oligosaccharide products.Through the combination of structural and biochemical analysis, we present new findings that provide novel insights into the degradation mechanism of chitosanase OU01.We have determined the crystal structure of Asp43/Ala mutant of OU01, and have trapped the hydrolyzed product of the reaction. This structure reveals the role of the general acid (Glu25) in catalysis. Two structural features about the mechanisms of the non-processive chitosanases are described for the first time. 1). Structural comparison reveals that the enzyme goes through an open–closed–open conformational transition upon substrate binding and product release; 2). polar residues constitute the substrate binding cleft. Additional site important for polymeric substrate recognition is identified and a three-step polymeric substrate recognition mechanism is proposed.Detailed substrate recognition mechanism is described for non-processive chitosanase for the first time.These findings provide new structural insights into the understanding of overall hydrolysis mechanism for non-processive chitosanase, and also will facilitate the design of new enzymes used for industrial purpose.
Keywords: Chitosan; Glycoside hydrolysis; Degradation; Mutagenesis; X-ray crystallography;