BBA - General Subjects (v.1860, #9)

Manganese ion concentration affects production of human core 3 O-glycan in Saccharomyces cerevisiae by Fumie Saito; Izumi Sakamoto; Akio Kanatani; Yasunori Chiba (1809-1820).
Production of various mucin-like glycoproteins could be useful for development of antibodies specific to disease-related glycoproteins as well as for the biosynthesis of clinically useful glycoproteins. A Saccharomyces cerevisiae strain capable of in vivo production of mucin-type core 1 structure (Galβ1-3GalNAcα1-O-Ser/Thr) has been reported, but a strain producing core 3 structure (GlcNAcβ1-3GalNAcα1-O-Ser/Thr) has not been constructed.To generate core 3-producing strain, genes encoding uridine diphosphate (UDP)-Gal-4-epimerase, UDP-GalNAc transporter, UDP-GlcNAc transporter, and two glycosyltransferases were integrated into the genome. A Mucin-1-derived acceptor peptide (MUC1ap) was expressed as an acceptor. The amount of the resulting modified peptide was analyzed by HPLC.Introduction of a codon-optimized UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyltransferase 6 (β3Gn-T6) gene yielded increases in β3Gn-T6 activity but did not alter the level of core 3 production. The highest in vitro activity of β3Gn-T6 was observed at Mn2 + concentrations of 10 mM and above. Supplementation of MnCl2 to the culture medium yielded increases of up to 25% in the accumulation of core 3 on the MUC1ap. The yeast invertase from the core 3-producing strain was less extensively N-glycosylated; however, it was partially restored by the addition of MnCl2 to the medium.Physiological Mn2 + concentration in S. cerevisiae was insufficient to facilitate optimal synthesis of core 3. Mn2 + supplementation led to up-regulation of reaction of glycosylation in the Golgi, resulting in increases of core 3 production.This study reveals that control of Mn2 + concentration is important for production of specific mammalian-type glycans in S. cerevisiae.
Keywords: Core 3-type O-glycan; Manganese ion; Glycan engineering; Yeast; Glycosyltransferase;

Hydration of proteins and nucleic acids: Advances in experiment and theory. A review by Lada Biedermannová; Bohdan Schneider (1821-1835).
Most biological processes involve water, and the interactions of biomolecules with water affect their structure, function and dynamics.This review summarizes the current knowledge of protein and nucleic acid interactions with water, with a special focus on the biomolecular hydration layer. Recent developments in both experimental and computational methods that can be applied to the study of hydration structure and dynamics are reviewed, including software tools for the prediction and characterization of hydration layer properties.In the last decade, important advances have been made in our understanding of the factors that determine how biomolecules and their aqueous environment influence each other. Both experimental and computational methods contributed to the gradually emerging consensus picture of biomolecular hydration.An improved knowledge of the structural and thermodynamic properties of the hydration layer will enable a detailed understanding of the various biological processes in which it is involved, with implications for a wide range of applications, including protein-structure prediction and structure-based drug design.
Keywords: Structural biology; Hydration; Biomolecular recognition; Molecular biophysics; Molecular modeling and simulation; Bioinformatics;

Biphasic reduction of histone H3 phosphorylation in response to N-nitroso compounds induced DNA damage by Kailin Chen; Shuilian Zhang; Xinxin Ke; Hongyan Qi; Jimin Shao; Jing Shen (1836-1844).
N-nitroso compounds (NOC) can cause cancers in a wide variety of animal species, and many of them are also potential human carcinogens. However, their underlying genotoxic mechanisms occurred within the context of chromatin, such as aberrant histone modifications, remained elusive.We investigated the dynamic landscapes of histone modifications after N-nitroso compound N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitroso-urea (MNU) exposure. Among the altered histone modifications, we also investigated the control mechanisms of histone H3 phosphorylation changes and its possible implications on transcriptional repression.Significantly, we find a specific biphasic reduction of histone H3 phosphorylation at serine 10 (H3S10ph) and serine 28 (H3S28ph), and a rapid decrease of histone H4 acetylation upon MNNG and MNU exposure. Further investigations reveal that the first hypophosphorylation of H3 occurs in a poly(ADP-ribosyl)ation enzyme PARP-1 (Poly(ADP-Ribose) Polymerase 1) dependent manner, whereas the second decline of H3 phosphorylation is at least partially under the control of histone kinase VRK1 (vaccinia-related kinase 1) and dependent on the tumor suppressor protein p53. In addition, DNA damage induced down-regulation of H3S10/S28 phosphorylation also functions in transcriptional repression of genes, such as cell-cycle regulators.Alkylating damage induced by NOC elicits a biphasic reduction of histone H3 phosphorylation with distinct control mechanisms, which is contributing to DNA damage responses such as the repair-facilitated transcriptional repression.Identification of the dynamic changes and underlying mechanisms of histone modifications upon NOC exposure would be of great help in understanding the epigenetic regulations of NOC induced DNA damage responses.
Keywords: N-nitroso compounds; Chemical carcinogen; DNA damage response; Epigenetics; Histone modifications;

Targeting soluble acid hydrolases to lysosomes requires the addition of mannose 6-phosphate residues on their N-glycans. This process is initiated by GlcNAc-1-phosphotransferase, a multi-subunit enzyme encoded by the GNPTAB and GNPTG genes. The GNPTAB gene products (the α and ß subunits) are responsible for recognition and catalysis of hydrolases whereas the GNPTG gene product (the γ subunit) enhances mannose phosphorylation of a subset of hydrolases. Here we identify and characterize a zebrafish gnptg insertional mutant and show that loss of the gamma subunit reduces mannose phosphorylation on a subset glycosidases but does not affect modification of several cathepsin proteases. We further show that glycosidases, but not cathepsins, are hypersecreted from gnptg −/− embryonic cells, as evidenced by reduced intracellular activity and increased circulating serum activity. The gnptg −/− embryos lack the gross morphological or craniofacial phenotypes shown in gnptab-deficient morphant embryos to result from altered cathepsin activity. Despite the lack of overt phenotypes, decreased fertilization and embryo survival were noted in mutants, suggesting that gnptg associated deposition of mannose 6-phosphate modified hydrolases into oocytes is important for early embryonic development. Collectively, these findings demonstrate that loss of the zebrafish GlcNAc-1-phosphotransferase γ subunit causes enzyme-specific effects on mannose phosphorylation. The finding that cathepsins are normally modified in gnptg −/− embryos is consistent with data from gnptab-deficient zebrafish suggesting these proteases are the key mediators of acute pathogenesis. This work also establishes a valuable new model that can be used to probe the functional relevance of GNPTG mutations in the context of a whole animal.Display Omitted
Keywords: Lysosomal hydrolase; Zebrafish; Cathepsin; Mannose phosphorylation;

Thioredoxin (Trx1) regulates CD4 membrane domain localization and is required for efficient CD4-dependent HIV-1 entry by Naazneen Moolla; Mark Killick; Maria Papathanasopoulos; Alexio Capovilla (1854-1863).
CD4 is a glycoprotein expressed on the surfaces of certain immune cells. On lymphocytes, an important function of CD4 is to co-engage Major Histocompatibility Complex (MHC) molecules with the T Cell Receptor (TCR), a process that is essential for antigen-specific activation of T cells. CD4 localizes dynamically into distinct membrane microdomains, an important feature of its immunoregulatory function that has also been shown to influence the efficiency of HIV replication. However, the mechanism by which CD4 localization is regulated and the biological significance of this is incompletely understood.In this study, we used confocal microscopy, density-gradient centrifugation and flow cytometry to analyze dynamic redox-dependent effects on CD4 membrane domain localization.Blocking cell surface redox exchanges with both a membrane-impermeable sulfhydryl blocker (DTNB) and specific antibody inhibitors of Thioredoxin-1 (Trx1) induces translocation of CD4 into detergent-resistant membrane domains (DRM). In contrast, Trx1 inactivation does not change the localization of the chemokine receptor CCR5, suggesting that this effect is targeted. Moreover, DTNB treatment and Trx1 depletion coincide with strong inhibition of CD4-dependent HIV entry, but only moderate reductions in the infectivity of a CD4-independent HIV pseudovirion.Changes in the extracellular redox environment, potentially mediated by allosteric consequences of functional disulfide bond oxidoreduction, may represent a signal for translocation of CD4 into DRM clusters, and this sequestration, another potential mechanism by which the anti-HIV effects of cell surface oxidoreductase inhibition are exerted.Extracellular redox conditions may regulate CD4 function by potentiating changes in its membrane domain localization.
Keywords: CD4; Redox; Thioredoxin; Lipid raft; HIV-1;

Disruption of the human COQ5-containing protein complex is associated with diminished coenzyme Q10 levels under two different conditions of mitochondrial energy deficiency by Hsiu-Chuan Yen; Yi-Chun Liu; Chia-Chi Kan; Hsing-Ju Wei; Szu-Hsien Lee; Yau-Huei Wei; Yu-Hsiu Feng; Chih-Wei Chen; Chin-Chang Huang (1864-1876).
The Coq protein complex assembled from several Coq proteins is critical for coenzyme Q6 (CoQ6) biosynthesis in yeast. Secondary CoQ10 deficiency is associated with mitochondrial DNA (mtDNA) mutations in patients. We previously demonstrated that carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) suppressed CoQ10 levels and COQ5 protein maturation in human 143B cells.This study explored the putative COQ protein complex in human cells through two-dimensional blue native-polyacrylamide gel electrophoresis and Western blotting to investigate its status in 143B cells after FCCP treatment and in cybrids harboring the mtDNA mutation that caused myoclonic epilepsy with ragged-red fibers (MERRF) syndrome. Ubiquinol-10 and ubiquinone-10 levels were detected by high-performance liquid chromatography. Mitochondrial energy status, mRNA levels of various PDSS and COQ genes, and protein levels of COQ5 and COQ9 in cybrids were examined.A high-molecular-weight protein complex containing COQ5, but not COQ9, in the mitochondria was identified and its level was suppressed by FCCP and in cybrids with MERRF mutation. That was associated with decreased mitochondrial membrane potential and mitochondrial ATP production. Total CoQ10 levels were decreased under both conditions, but the ubiquinol-10:ubiquinone-10 ratio was increased in mutant cybrids. The expression of COQ5 was increased but COQ5 protein maturation was suppressed in the mutant cybrids.A novel COQ5-containing protein complex was discovered in human cells. Its destabilization was associated with reduced CoQ10 levels and mitochondrial energy deficiency in human cells treated with FCCP or exhibiting MERRF mutation.The findings elucidate a possible mechanism for mitochondrial dysfunction-induced CoQ10 deficiency in human cells.
Keywords: High-molecular-weight protein complex; Human COQ5; Human COQ9; Chemical uncoupler; mtDNA mutation; Secondary coenzyme Q10 deficiency;

Monitoring drug induced apoptosis and treatment sensitivity in non-small cell lung carcinoma using dielectrophoresis by Rajeshwari Taruvai Kalyana Kumar; Shanshan Liu; John D. Minna; Shalini Prasad (1877-1883).
Non-invasive real time methods for characterizing biomolecular events that contribute towards apoptotic kinetics would be of significant importance in the field of cancer biology. Effective drug-induced apoptosis is an important factor for establishing the relationship between cancer genetics and treatment sensitivity. The objective of this study was to develop a non-invasive technique to characterize cancer cells that are undergoing drug-induced apoptosis. We used dielectrophoresis to determine apoptotic cells as early as 2 h post drug treatment as compared to 24 h with standard flow cytometry method using non-small cell lung cancer (NSCLC) adenocarcinoma cell line (HCC1833) as a study model. Our studies have shown significant differences in apoptotic cells by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine (PS) on the extracellular surface when the cells where treated with a potent Bcl-2 family inhibitor drug (ABT-263). Time lapse dielectrophoretic studies were performed over 24 h period after exposure to ABT-263 at clinically relevant concentrations. The dielectrophoretic studies were compared to Annexin-V FITC flow assay for the detection of PS in mid-stage apoptosis using flow cytometry. As a result of physical and biochemical changes, inherent dielectric properties of cells undergoing varying stages of apoptosis showed amplified changes in their cytoplasmic and membrane capacitance. In addition, zeta potential of these fixed isolated cells was measured to obtain direct correlation to biomolecular events.Display Omitted
Keywords: Non invasive electrokinetics; Apoptosis monitoring; Dielectrophoresis; Non-small cell lung cancer;

Secoisolariciresinol diglucoside (SDG), the main lignan in whole grain flaxseed, is a potent antioxidant and free radical scavenger with known radioprotective properties. However, the exact mechanism of SDG radioprotection is not well understood. The current study identified a novel mechanism of DNA radioprotection by SDG in physiological solutions by scavenging active chlorine species (ACS) and reducing chlorinated nucleobases.The ACS scavenging activity of SDG was determined using two highly specific fluoroprobes: hypochlorite-specific 3′-(p-aminophenyl) fluorescein (APF) and hydroxyl radical-sensitive 3′-(p-hydroxyphenyl) fluorescein (HPF). Dopamine, an SDG structural analog, was used for proton 1H NMR studies to trap primary ACS radicals. Taurine N-chlorination was determined to demonstrate radiation-induced generation of hypochlorite, a secondary ACS. DNA protection was assessed by determining the extent of DNA fragmentation and plasmid DNA relaxation following exposure to ClO and radiation. Purine base chlorination by ClO and γ-radiation was determined by using 2-aminopurine (2-AP), a fluorescent analog of 6-aminopurine. Results: Chloride anions (Cl) consumed > 90% of hydroxyl radicals in physiological solutions produced by γ-radiation resulting in ACS formation, which was detected by 1H NMR. Importantly, SDG scavenged hypochlorite- and γ–radiation-induced ACS. In addition, SDG blunted ACS-induced fragmentation of calf thymus DNA and plasmid DNA relaxation. SDG treatment before or after ACS exposure decreased the ClO or γ-radiation-induced chlorination of 2-AP. Exposure to γ-radiation resulted in increased taurine chlorination, indicative of ClO generation. NMR studies revealed formation of primary ACS radicals (chlorine atoms (Cl) and dichloro radical anions (Cl2¯ )), which were trapped by SDG and its structural analog dopamine.We demonstrate that γ-radiation induces the generation of ACS in physiological solutions. SDG treatment scavenged ACS and prevented ACS-induced DNA damage and chlorination of 2-aminopurine. This study identified a novel and unique mechanism of SDG radioprotection, through ACS scavenging, and supports the potential usefulness of SDG as a radioprotector and mitigator for radiation exposure as part of cancer therapy or accidental exposure.
Keywords: Active chlorine species; 2-Aminopurine; Chlorine atoms; Dichloro radical anion; DNA fragmentation; Hydroxyl radical; Hypochlorite ion; γ-Radiation; Mitigation; Radicals; Radioprotection; Secoisolariciresinol diglucoside; Taurine chloramine;

A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection by Andrea Troupin; Berlin Londono-Renteria; Michael J. Conway; Erin Cloherty; Samuel Jameson; Stephen Higgs; Dana L. Vanlandingham; Erol Fikrig; Tonya M. Colpitts (1898-1909).
Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant human disease and mortality in the tropics and subtropics. By examining the effects of virus infection on gene expression, and interactions between virus and vector, new targets for prevention of infection and novel treatments may be identified in mosquitoes. We previously performed a microarray analysis of the Aedes aegypti transcriptome during infection with DENV and found that mosquito ubiquitin protein Ub3881 (AAEL003881) was specifically and highly down-regulated. Ubiquitin proteins have multiple functions in insects, including marking proteins for proteasomal degradation, regulating apoptosis and mediating innate immune signaling.We used qRT-PCR to quantify gene expression and infection, and RNAi to reduce Ub3881 expression. Mosquitoes were infected with DENV through blood feeding. We transfected DENV protein expression constructs to examine the effect of Ub3881 on protein degradation. We used site-directed mutagenesis and transfection to determine what amino acids are involved in Ub3881-mediated protein degradation. Immunofluorescence, Co-immunoprecipitation and Western blotting were used to examine protein interactions and co-localization.The overexpression of Ub3881, but not related ubiquitin proteins, decreased DENV infection in mosquito cells and live Ae. aegypti. The Ub3881 protein was demonstrated to be involved in DENV envelope protein degradation and reduce the number of infectious virions released.We conclude that Ub3881 has several antiviral functions in the mosquito, including specific viral protein degradation.Our data highlights Ub3881 as a target for future DENV prevention strategies in the mosquito transmission vector.Display Omitted
Keywords: Dengue virus; Mosquito transmission; Ubiquitin; Protein degradation; Arbovirus;

Unification of [FeFe]-hydrogenases into three structural and functional groups by Saroj Poudel; Monika Tokmina-Lukaszewska; Daniel R. Colman; Mohammed Refai; Gerrit J. Schut; Paul W. King; Pin-Ching Maness; Michael W.W. Adams; John W. Peters; Brian Bothner; Eric S. Boyd (1910-1921).
[FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2.To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods.HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation.These results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA.This classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes.
Keywords: [FeFe]-hydrogenase; Hydrogen; Electron bifurcation; Post-translational modification; Regulation; Bioinformatics;

A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers by Deo R. Singh; Elena B. Pasquale; Kalina Hristova (1922-1928).
The EphA2 receptor tyrosine kinase is known to promote cancer cell malignancy in the absence of activation by ephrin ligands. This behavior depends on high EphA2 phosphorylation on Ser897 and low tyrosine phosphorylation, resulting in increased cell migration and invasiveness. We have previously shown that EphA2 forms dimers in the absence of ephrin ligand binding, and that dimerization of unliganded EphA2 can decrease EphA2 Ser897 phosphorylation. We have also identified a small peptide called YSA, which binds EphA2 and competes with the naturally occurring ephrin ligands.Here, we investigate the effect of YSA on EphA2 dimer stability and EphA2 function using quantitative FRET techniques, Western blotting, and cell motility assays.We find that the YSA peptide stabilizes the EphA2 dimer, increases EphA2 Tyr phosphorylation, and decreases both Ser897 phosphorylation and cell migration.The experiments demonstrate that the small peptide ligand YSA reduces EphA2 Ser897 pro-tumorigenic signaling by stabilizing the EphA2 dimer.This work is a proof-of-principle demonstration that EphA2 homointeractions in the plasma membrane can be pharmacologically modulated to decrease the pro-tumorigenic signaling of the receptor.

Tetracycline-loaded calcium phosphate nanoparticle (Tet-CPNP): Rejuvenation of an obsolete antibiotic to further action by Riya Mukherjee; Mousumi Patra; Debanjan Dutta; Milon Banik; Tarakdas Basu (1929-1941).
Increasing resistance in bacteria towards antibiotics has made it imperative to research on their revitalization to combat infectious diseases. This study dealt with synthesis of a nano-form of the antibiotic tetracycline, its characterization and potency of killing different multi-drug resistant diarrhea-causing bacteria.Nano-formulation was done by loading tetracycline within biocompatible calcium phosphate nanoparticle. The synthesized tetracycline-loaded calcium phosphate nanoparticle (Tet-CPNP) was characterized by the techniques like TEM, DLS, EDS, FTIR, spectrofluorimetry and dialysis. Bactericidal activity of nano-particulate tetracycline was investigated by agar plating, spectrophotometry, phase contrast-fluorescence-atomic force microscopy and flow cytometry techniques.The Tet-CPNPs were 8 ± 5 nm in size and nearly spherical in shape, efficiency of tetracycline loading in CPNP was about 20% and the release of antibiotic from Tet-CPNPs was sustainable during 7 days. Minimum inhibitory concentration (MIC) of Tet-CPNP on multiple antibiotic (including tetracycline) resistant bacteria like Escherichia coli, Salmonella kentuckey and Shigella flexneri was in the range of 20–40 μg/ml, whereas MIC of free tetracycline was in the range of 150–180 μg/ml. NP-mediated cell filamentation and cell membrane disintegration caused cell killing. Moreover, death of Shigella-infected Zebra fish larvae was stalled by Tet-CPNP treatment. CPNP itself had no toxic effect on bacteria as well as on Zebra fish.Our nano-formulation of tetracycline might reclaim a nearly obsolete antibiotic to further potential function.Such a study on revival of an old, cheap, broad-spectrum antibiotic to further action is highly beneficial to developing countries with limited health care budgets.Display Omitted
Keywords: Tetracycline; Calcium phosphate nanoparticle; MIC; Diarrhea-causing multi-drug resistant bacteria; Zebra fish;

Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans by Szilvia Barkó; Dávid Szatmári; Emőke Bódis; Katalin Türmer; Zoltán Ujfalusi; David Popp; Robert C. Robinson; Miklós Nyitrai (1942-1952).
Weil's syndrome is caused by Leptospira interrogans infections, a Gram negative bacterium with a distinct thin corkscrew cell shape. The molecular basis for this unusual morphology is unknown. In many bacteria, cell wall synthesis is orchestrated by the actin homolog, MreB.Here we have identified the MreB within the L. interrogans genome and expressed the His-tagged protein product of the synthesized gene (Li-MreB) in Escherichia coli. Li-MreB did not purify under standard nucleotide-free conditions used for MreBs from other species, requiring the continual presence of ATP to remain soluble. Covalent modification of Li-MreB free thiols with Alexa488 produced a fluorescent version of Li-MreB.We developed native and denaturing/refolding purification schemes for Li-MreB. The purified product was shown to assemble and disassemble in MgCl2 and KCl dependent manners, as monitored by light scattering and sedimentation studies. The fluorescence spectrum of labeled Li-MreB-Alexa488 showed cation-induced changes in line with an activation process followed by a polymerization phase. The resulting filaments appeared as bundles and sheets under the fluorescence microscope. Finally, since the Li-MreB polymerization was cation dependent, we developed a simple method to measure monovalent cation concentrations within a test case prokaryote, E. coli.We have identified and initially characterized the cation-dependent polymerization properties of a novel MreB from a non-rod shaped bacterium and developed a method to measure cation concentrations within prokaryotes.General significance.This initial characterization of Li-MreB will enable future structural determination of the MreB filament from this corkscrew-shaped bacterium.
Keywords: MreB; Bacterial actin; Cytoskeleton; Polymerization; Fluorescence microscopy;

Cell stiffness determined by atomic force microscopy and its correlation with cell motility by Qing Luo; Dongdong Kuang; Bingyu Zhang; Guanbin Song (1953-1960).
Cell stiffness is a crucial mechanical property that is closely related to cell motility. AFM is the most prevalent method used to determine cell stiffness by the quantitative parameter designated as Young's modulus. Young's modulus is regarded as a biomarker of cell motility, especially in estimating the metastasis of cancer cells, because in recent years, it has been repeatedly shown that cancerous cells are softer than their benign counterparts. However, some conflicting evidence has shown that cells with higher motility are sometimes stiffer than their counterparts. Thus, the correlation between cell stiffness and motility remains a matter of debate.In this review, we first summarize the reports on correlations between cell motility and stiffness determined by AFM and then discuss the major determinants of AFM-determined cell stiffness with a focus on the cytoskeleton, nuclear stiffness and methodological issues. Last, we propose a possible correlation between cell stiffness and motility and the possible explanations for the conflicting evidence.The AFM-determined Young's modulus is greatly affected by the characteristics of the cytoskeleton, as well as the procedures and parameters used in detection. Young's modulus is a reliable biomarker for the characterization of metastasis; however, reliability is questioned in the evaluation of pharmacologically or genetically modified motility.This review provides an overview of the current understanding of the correlation between AFM-determined cell stiffness and motility, the determinants of this detecting method, as well as clues to optimize detecting parameters.
Keywords: Atomic force microscopy; Motility; Stiffness; Cytoskeleton; Nuclear stiffness; Young's modulus;

Smoothened-independent activation of hedgehog signaling by rearranged during transfection promotes neuroblastoma cell proliferation and tumor growth by Hongfeng Ruan; Huan Luo; Jirong Wang; Xing Ji; Zhongmiao Zhang; Junsong Wu; Xianning Zhang; Ximei Wu (1961-1972).
Rearranged during transfection (RET) proto-oncogene encodes a receptor tyrosine kinase for glial cell line-derived neurotrophic factor (GDNF) signaling, and high RET expression is closely related to the tumorigenesis and malignancy of neuroblastoma(NB).We have investigated whether RET signals through hedgehog (HH) pathway in NB cell proliferation and tumor growth by in vitro cell culture and in vivo xenograft approaches.The key members of both GDNF/RET and HH/GLI pathways are expressed in NB cell lines to different extents. Knockdown of RET in NB cells significantly attenuates the activity of HH signaling, whereas overexpression of RET robustly enhances the output of transcriptional activation by HH. Likewise, activation of RET by GDNF induces HH signaling, whereas knockdown of RET attenuates both basal and GDNF-induced activities of HH signaling. Moreover, protein kinase B lies on the downstream of GDNF/RET signaling module to inhibit the GSK3β, resulting in activation of HH signaling. Furthermore, either knockdown of RET by shRNA or inhibition of HH pathway by cyclopamine attenuates not only basal but also GDNF-induced proliferation of SH-SY5Y cells, and knockdown of either RET or smoothened in SH-SY5Y cell xenografts significantly attenuated the tumor growth. Finally, inhibition of HH signaling by GLI1 and GLI2 inhibitor, Gant61, reduces not only basal but also RET-induced proliferation of SH-SY5Y cells and outgrowth of xenografts.GDNF/RET/AKT/GSK3β signaling module activates HH pathway to stimulate NB cells proliferation and tumor outgrowth.Targeting HH pathway is a rational approach for therapeutic intervention of NB with high RET expression.
Keywords: GDNF; RET; Hedgehog; Neuroblastoma;

Leishmania donovani Aurora kinase: A promising therapeutic target against visceral leishmaniasis by Rudra Chhajer; Anirban Bhattacharyya; Nicky Didwania; Md Shadab; Nirupam Das; Partha Palit; Tushar Vaidya; Nahid Ali (1973-1988).
Aurora kinases are key mitotic kinases executing multiple aspects of eukaryotic cell-division. The apicomplexan homologs being essential for survival, suggest that the Leishmania homolog, annotated LdAIRK, may be equally important.Bioinformatics, stage-specific immunofluorescence microscopy, immunoblotting, RT-PCR, molecular docking, in-vitro kinase assay, anti-leishmanial activity assays, flow cytometry, fluorescence microscopy.Ldairk expression is seen to vary as the cell-cycle progresses from G1 through S and finally G2M and cytokinesis. Kinetic studies demonstrate their enzymatic activity exhibiting a Km and Vmax of 6.12 μM and 82.9pmoles·min− 1mg− 1 respectively against ATP using recombinant Leishmania donovani H3, its physiological substrate. Due to the failure of LdAIRK −/+ knock-out parasites to survive, we adopted a chemical knock-down approach. Based on the conservation of key active site residues, three mammalian Aurora kinase inhibitors were investigated to evaluate their potential as inhibitors of LdAIRK activity. Interestingly, the cell-cycle progressed unhindered, despite treatment with GSK-1070916 or Barasertib, inhibitors with greater potencies for the ATP-binding pocket compared to Hesperadin, which at nanomolar concentrations, severely compromised viability at IC50s 105.9 and 36.4 nM for promastigotes and amastigotes, respectively. Cell-cycle and morphological studies implicated their role in both mitosis and cytokinesis.We identified an Aurora kinase homolog in L. donovani implicated in cell-cycle progression, whose inhibition led to aberrant changes in cell-cycle progression and reduced viability.Human homologs being actively pursued drug targets and the observations with LdAIRK in both promastigotes and amastigotes suggest their potential as therapeutic-targets. Importantly, our results encourage the exploration of other proteins identified herein as potential novel drug targets.
Keywords: Aurora kinase; Cytokinesis; Hesperadin; Therapeutic target; Cell-cycle; Leishmania donovani;

Dopamine replacement therapy by its precursor, L-3.4-dihydroxyphenylalanine (L-DOPA), has been the treatment of choice for Parkinson's disease. However, the possible contributory effect of L-DOPA therapy on the progression of Parkinson's disease mediated by the L-DOPA-induced toxic metabolites remains elusive.Prolong use of L-DOPA leads to behavioral impediments and instigate the generation of several toxic metabolites. One such metabolite is homocysteine, the level of which increases in the plasma of Parkinson's disease patients undergoing L-DOPA therapy, as well as in brain of animal models of the disease. In concoction with parkinsonian neurotoxins, Hcy exaggerates dopaminergic neurodegeneration, while its intranigral infusion has been demonstrated to decrease the dopamine level as well as causes dopaminergic neurodegeneration. Therefore, it can be propounded that elevated level of Hcy (hyperhomocysteinemia) is one of the underlying causes of L-DOPA-induced side-effects and aggravates the progressive nature of Parkinson's disease, which has been focused here. We have provided a conjectural discussion on the involvement of Hcy in L-DOPA-induced dyskinesia in Parkinson's disease.Hyperhomocysteinemia as a result of prolonged L-DOPA therapy is the emerging cause of L-DOPA-induced behavioral abnormalities and progressive nature of Parkinson's disease.This review highlights that hyperhomocysteinemia could be a putative contributor of the side-effects of chronic L-DOPA therapy because of its neurotoxic potency.
Keywords: Homocysteine; Neurotoxicity; Dopaminergic neurons; Oxidative stress; Catechol O-methyl transferase; Dyskinesia;

PI3K pathway is involved in ERK signaling cascade activation by histamine H2R agonist in HEK293T cells by Natalia Alonso; Antonela Diaz Nebreda; Federico Monczor; J. Silvio Gutkind; Carlos Davio; Natalia Fernandez; Carina Shayo (1998-2007).
Histamine, through histamine H2 receptor (H2R), modulates different biological processes, involving the modulation of PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways. Many evidences have demonstrated the existence and importance of the crossregulation between these two signaling pathways. The aim of the present work was to determine the molecular mechanisms leading to PI3K and ERK pathways modulation induced by the H2R agonist amthamine and to evaluate the possible interplay between them.Phosphorylation levels of ERK and Akt were examined by Western blot in HEK293T cells expressing the human H2R, in the presence of H2R agonist and dominant negative mutants or pharmacological inhibitors of different proteins/pathways. Transcriptional activity assays were assessed to determine SRE activity. Amthamine-mediated cellular proliferation was investigated in MA-10A cells in the presence of PI3K inhibitor.H2R agonist inhibits PI3K/Akt/mTOR and stimulates Ras/MEK/ERK pathways. Moreover, PI3K/Akt/mTOR signaling inhibition is necessary to achieve H2R mediated ERK activation. In the presence of a constitutive active mutant of Akt, amthamine is not able to mediate ERK activation. This crosstalk affects classical ERK downstream targets such as Elk1 phosphorylation and the transcriptional activity of the SRE, classically associated to proliferation. We further demonstrate that amthamine-induced proliferation in Leydig MA-10 tumor cells, is enhanced by LY294002, a PI3K inhibitor.These results describe a crosstalk between PI3K/AKT/mTOR and Ras/MEK/ERK pathways induced by H2R stimulation with implications in cell proliferation.This work indicates that the modulation of PI3K/AKT/mTOR pathway by H2R in turn regulates Ras/MEK/ERK activation conditioning the proliferative capacity of the cells.
Keywords: Histamine H2 receptor; ERK; PI3K; Cell proliferation; Cell signaling;

Transplantation of islets of Langerhans is regarded as a promising therapy for type 1 diabetes. A large number of β-cells are required for the treatment of human type 1 diabetes. Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, have been considered as new sources for cell replacement therapy.Cell aggregates were prepared from human iPS cells using agarose microwell plates and differentiated into pancreatic endocrine cells by changing the culture media with different additives.After 20 days of culture, approximately 30% of cells in aggregates were positive for C-peptide. After another 14 days in culture, the cells gained an ability to alter C-peptide release in response to changes in the glucose concentration.Uniform aggregates of human iPSCs were easily prepared on agarose microwell plates and efficiently differentiated into the pancreatic endocrine lineage. Thus, aggregate culture is a suitable method for preparing islet-like aggregates from human iPSCs.Our results indicate that the microwell plate is suitable for scaling up the preparation of pancreatic endocrine cells from human iPS cells in a robotic system.
Keywords: Induced pluripotent stem cell; Islet transplantation; Differentiation; Regenerative medicine;

O-GlcNAcylation is a key modulator of skeletal muscle sarcomeric morphometry associated to modulation of protein–protein interactions by Matthias Lambert; Elodie Richard; Sophie Duban-Deweer; Frederic Krzewinski; Barbara Deracinois; Erwan Dupont; Bruno Bastide; Caroline Cieniewski-Bernard (2017-2030).
The sarcomere structure of skeletal muscle is determined through multiple protein–protein interactions within an intricate sarcomeric cytoskeleton network. The molecular mechanisms involved in the regulation of this sarcomeric organization, essential to muscle function, remain unclear. O-GlcNAcylation, a post-translational modification modifying several key structural proteins and previously described as a modulator of the contractile activity, was never considered to date in the sarcomeric organization.C2C12 skeletal myotubes were treated with Thiamet-G (OGA inhibitor) in order to increase the global O-GlcNAcylation level.Our data clearly showed a modulation of the O-GlcNAc level more sensitive and dynamic in the myofilament-enriched fraction than total proteome. This fine O-GlcNAc level modulation was closely related to changes of the sarcomeric morphometry. Indeed, the dark-band and M-line widths increased, while the I-band width and the sarcomere length decreased according to the myofilament O-GlcNAc level. Some structural proteins of the sarcomere such as desmin, αB-crystallin, α-actinin, moesin and filamin-C have been identified within modulated protein complexes through O-GlcNAc level variations. Their interactions seemed to be changed, especially for desmin and αB-crystallin.For the first time, our findings clearly demonstrate that O-GlcNAcylation, through dynamic regulations of the structural interactome, could be an important modulator of the sarcomeric structure and may provide new insights in the understanding of molecular mechanisms of neuromuscular diseases characterized by a disorganization of the sarcomeric structure.In the present study, we demonstrated a role of O-GlcNAcylation in the sarcomeric structure modulation.
Keywords: Sarcomere structure; Skeletal muscle cells; O-GlcNAcylation; Protein–protein interactions; Desmin; αB-crystallin;

En route from artificial to natural: Evaluation of inhibitors of mannose-specific adhesion of E. coli under flow by Leonhard Möckl; Claudia Fessele; Guillaume Despras; Christoph Bräuchle; Thisbe K. Lindhorst (2031-2036).
We investigated the properties of six Escherichia coli adhesion inhibitors under static and under flow conditions. On mannan-covered model substrates and under static conditions, all inhibitors were able to almost completely abolish lectin-mediated E. coli adhesion. On a monolayer of living human microvascular endothelial cells (HMEC-1), the inhibitors reduced adhesion under static conditions as well, but a large fraction of bacteria still managed to adhere even at highest inhibitor concentrations. In contrast, under flow conditions E. coli did not exhibit any adhesion to HMEC-1 not even at inhibitor concentrations where significant adhesion was detected under static conditions. This indicates that the presence of shear stress strongly affects inhibitor properties and must be taken into account when evaluating the potency of bacterial adhesion inhibitors.
Keywords: Bacterial adhesion; Antiadhesion therapy; Carbohydrate recognition; Flow conditions; Shear stress;

Mitogen-activated protein kinases as key players in osmotic stress signaling by Xiangyu Zhou; Isao Naguro; Hidenori Ichijo; Kengo Watanabe (2037-2052).
Osmotic stress arises from the difference between intracellular and extracellular osmolality. It induces cell swelling or shrinkage as a consequence of water influx or efflux, which threatens cellular activities. Mitogen-activated protein kinases (MAPKs) play central roles in signaling pathways in osmotic stress responses, including the regulation of intracellular levels of inorganic ions and organic osmolytes.The present review summarizes the cellular osmotic stress response and the function and regulation of the vertebrate MAPK signaling pathways involved. We also describe recent findings regarding apoptosis signal-regulating kinase 3 (ASK3), a MAP3K member, to demonstrate its regulatory effects on signaling molecules beyond MAPKs.MAPKs are rapidly activated by osmotic stress and have diverse roles, such as cell volume regulation, gene expression, and cell survival/death. There is significant cell type specificity in the function and regulation of MAPKs. Based on its activity change during osmotic stress and its regulation of the WNK1-SPAK/OSR1 pathway, ASK3 is expected to play important roles in osmosensing mechanisms and cellular functions related to osmoregulation.MAPKs are essential for various cellular responses to osmotic stress; thus, the identification of the upstream regulators of MAPK pathways will provide valuable clues regarding the cellular osmosensing mechanism, which remains elusive in mammals. The elucidation of in vivo MAPK functions is also important because osmotic stress in physiological and pathophysiological conditions often results from changes in the intracellular osmolality. These studies potentially contribute to the establishment of therapeutic strategies against diseases that accompany osmotic perturbation.
Keywords: Mitogen-activated protein kinase; Osmotic stress; Regulatory volume decrease; Regulatory volume increase; Apoptosis signal-regulating kinase 3; With-no-lysine kinase;

Molecular interaction of δ-conopeptide EVIA with voltage-gated Na+ channels by Daniel Tietze; Enrico Leipold; Pascal Heimer; Miriam Böhm; Wadim Winschel; Diana Imhof; Stefan H. Heinemann; Alesia A. Tietze (2053-2063).
For a large number of conopeptides basic knowledge related to structure-activity relationships is unavailable although such information is indispensable with respect to drug development and their use as drug leads.A combined experimental and theoretical approach employing electrophysiology and molecular modeling was applied for identifying the conopeptide δ-EVIA binding site at voltage-gated Na+ channels and to gain insight into the toxin's mode of action.Conopeptide δ-EVIA was synthesized and its structure was re-determined by NMR spectroscopy for molecular docking studies. Molecular docking and molecular dynamics simulation studies were performed involving the domain IV voltage sensor in a resting conformation and part of the domain I S5 transmembrane segment. Molecular modeling was stimulated by functional studies, which demonstrated the importance of domains I and IV of the neuronal NaV1.7 channel for toxin action.δ-EVIA shares its binding epitope with other voltage-sensor toxins, such as the conotoxin δ-SVIE and various scorpion α-toxins. In contrast to previous in silico toxin binding studies, we present here in silico binding studies of a voltage-sensor toxin including the entire toxin binding site comprising the resting domain IV voltage sensor and S5 of domain I.The prototypical voltage-sensor toxin δ-EVIA is suited for the elucidation of its binding epitope; in-depth analysis of its interaction with the channel target yields information on the mode of action and might also help to unravel the mechanism of voltage-dependent channel gating and coupling of activation and inactivation.Display Omitted
Keywords: Conotoxin; δ-EVIA; NMR structural analysis; Voltage-sensor toxin; Molecular dynamics simulations;