BBA - General Subjects (v.1850, #11)

Role of P2X7 receptor in Clostridium perfringens beta-toxin-mediated cellular injury by Masahiro Nagahama; Soshi Seike; Hidenori Shirai; Teruhisa Takagishi; Keiko Kobayashi; Masaya Takehara; Jun Sakurai (2159-2167).
Clostridium perfringens beta-toxin is a pore-forming toxin (PFT) and an important agent of necrotic enteritis and enterotoxemia. We recently reported that beta-toxin strongly induced cell death in THP-1 cells via the formation of oligomers. We here describe that the P2X7 receptor, which is an ATP receptor, interacts with beta-toxin.We tested the role of P2X7 receptor in beta-toxin-induced toxicity using specific inhibitors, knockdown of receptor, expression of the receptor and interaction by dot-blot assay. The potency of P2X7 receptor was further determined using an in vivo mouse model.Selective P2X7 receptor antagonists (oxidized ATP (o-ATP), oxidized ADP, and Brilliant Blue G (BBG)) inhibited beta-toxin-induced cytotoxicity in THP-1 cells. o-ATP also blocked the binding of beta-toxin to cells. The P2X7 receptor and beta-toxin oligomer were localized in the lipid rafts of THP-1 cells. siRNA for the P2X7 receptor inhibited toxin-induced cytotoxicity and binding of the toxin. In contrast, the siRNA knockdown of P2Y2 or P2Y6 had no effect on beta-toxin-induced cytotoxicity. The addition of beta-toxin to P2X7-transfected HEK-293 cells resulted in binding of beta-toxin oligomer. Moreover, beta-toxin specifically bound to immobilized P2X7 receptors in vitro and colocalized with the P2X7 receptor on the THP-1 cell surface. Furthermore, beta-toxin-induced lethality in mice was blocked by the preadministration of BBG.The results of this study indicate that the P2X7 receptor plays a role in beta-toxin-mediated cellular injury.P2X7 receptor is a potential target for the treatment of C. perfringens type C infection.
Keywords: C. perfringens beta-toxin; P2X7 receptor; Pore-forming toxin; Oligomer formation;

Distinctive malfunctions of calmodulin mutations associated with heart RyR2-mediated arrhythmic disease by Vyronia Vassilakopoulou; Brian L. Calver; Angelos Thanassoulas; Konrad Beck; Handan Hu; Luke Buntwal; Adrian Smith; Maria Theodoridou; Junaid Kashir; Lynda Blayney; Evangelia Livaniou; George Nounesis; F. Anthony Lai; Michail Nomikos (2168-2176).
Calmodulin (CaM) is a cytoplasmic calcium sensor that interacts with the cardiac ryanodine receptor (RyR2), a large Ca2 + channel complex that mediates Ca2 + efflux from the sarcoplasmic reticulum (SR) to activate cardiac muscle contraction. Direct CaM association with RyR2 is an important physiological regulator of cardiac muscle excitation–contraction coupling and defective CaM–RyR2 protein interaction has been reported in cases of heart failure. Recent genetic studies have identified CaM missense mutations in patients with a history of severe cardiac arrhythmogenic disorders that present divergent clinical features, including catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT syndrome (LQTS) and idiopathic ventricular fibrillation (IVF). Herein, we describe how two CPVT- (N54I & N98S) and three LQTS-associated (D96V, D130G & F142L) CaM mutations result in alteration of their biochemical and biophysical properties. Ca2 +-binding studies indicate that the CPVT-associated CaM mutations, N54I & N98S, exhibit the same or a 3-fold reduced Ca2 +-binding affinity, respectively, versus wild-type CaM, whereas the LQTS-associated CaM mutants, D96V, D130G & F142L, display more profoundly reduced Ca2 +-binding affinity. In contrast, all five CaM mutations confer a disparate RyR2 interaction and modulation of [3H]ryanodine binding to RyR2, regardless of CPVT or LQTS association. Our findings suggest that the clinical presentation of CPVT or LQTS associated with these five CaM mutations may involve both altered intrinsic Ca2 +-binding as well as defective interaction with RyR2.
Keywords: Calmodulin; Calcium; Ryanodine receptor; RyR2 calcium release channel; Cardiac disease;

Using an in vitro model to study oxidised protein accumulation in ageing fibroblasts by Nae Shiozawa-West; Rachael A. Dunlop; Kenneth J. Rodgers (2177-2184).
The accumulation of oxidised proteins in ageing cells and tissues results from an increase in oxidant damage coupled with impaired degradation of the damaged proteins. Heat Shock Proteins (HSP) and other chaperones are required to recognise damaged proteins and transport them to the lysosomal and proteasomal degradation pathways. How these systems fail in ageing cells is not clear.We monitor oxidised protein accumulation, the activity of the proteasome and lysosomal proteases, and HSP levels in MRC-5 fibroblasts throughout their mitotic lifespan. We then use a novel in vitro cell culture model to experimentally generate oxidised proteins in young and old MRC-5 fibroblasts and compare their rates of degradation and changes in the key pathways involved in oxidised protein removal.We show that the activity of the proteasome and some lysosomal enzymes decreases with ageing in MRC-5 cells as do levels of HSP70 but this is not associated with an accumulation of oxidised proteins which only occurs as cells closely approach post-mitotic senescence. Old cells are unable to degrade experimentally generated oxidised proteins as efficiently as young cells. Exposure to mild heat stress however increases the efficiency of oxidised protein degradation by young cells and increases levels of HSP70.Our results highlight the importance of the HSP/chaperone system in oxidised protein metabolism, particularly in ageing cells.These data might have implications for the development of therapies for pathologies associated with protein accumulation and suggest that the HSP/chaperone system would be an important target.
Keywords: Oxidised proteins; Ageing; DOPA; Proteasome; Lysosomes;

Omega-3 DHA- and EPA–dopamine conjugates induce PPARγ-dependent breast cancer cell death through autophagy and apoptosis by Daniela Rovito; Cinzia Giordano; Pierluigi Plastina; Ines Barone; Francesca De Amicis; Loredana Mauro; Pietro Rizza; Marilena Lanzino; Stefania Catalano; Daniela Bonofiglio; Sebastiano Andò (2185-2195).
The omega-3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) may form conjugates with amines that have potential health benefits against common diseases including cancers. Here we synthesized DHA-dopamine (DHADA) and EPA–dopamine (EPADA) conjugates and studied their biological effects on different breast cancer cell lines.MTT assays indicated that increasing concentrations of DHADA and EPADA significantly affected viability in MCF-7, SKBR3 and MDA-MB-231 breast cancer cells, whereas no effect was observed in MCF-10A non-tumorigenic epithelial breast cells. DHADA and EPADA enhanced Beclin-1 expression, as evidenced by immunoblotting, real-time-PCR and functional analyses. Chromatin Immunoprecipitation (ChIP) and Re-ChIP assays revealed that both compounds induced recruitment of Peroxisome-Proliferator-Activated-Receptor gamma (PPARγ) and RNA Polymerase-II at the Retinoic-X-Receptor binding region on Beclin-1 promoter. Moreover, both compounds enhanced autophagosome formation, evaluated by LC-3 and monodansylcadaverine labeling, that was prevented by the PPARγ antagonist GW9662, addressing the direct involvement of PPARγ. Noteworthy, long-term treatment with DHADA and EPADA caused the blockade of autophagic flux followed by apoptotic cell death as evidenced by PARP cleavage and DNA fragmentation in all breast cancer cells.We have provided new insights into the molecular mechanism through which PPARγ, as a central molecule in the cross talk between autophagy and apoptosis, mediates DHADA- and EPADA-induced cell death in breast cancer cells.Our findings suggest that omega-3 DHADA- and EPADA activation of PPARγ may assume biological relevance in setting novel adjuvant therapeutic interventions in breast carcinoma.
Keywords: Apoptosis; Autophagy; Beclin-1; Breast cancer; Omega-3 polyunsaturated fatty acid conjugates; Peroxisome Proliferator-Activated Receptor gamma;

The insulin signaling pathway functions as a major regulator of many metabolic and cellular functions, and has been shown to be reversibly suppressed in many species during hibernation. This study characterized the regulation of PTEN phosphatase, a negative regulator of the insulin receptor network, over the torpor–arousal cycle of hibernation in the skeletal muscle of Ictidomys tridecemlineatus.Western blotting and RT-PCR were used to analyze post-translational and transcriptional regulations of PTEN respectively. Enzymatic activities were determined by the malachite green assay, while protein stability was assessed the using pulse-proteolysis method.During torpor, the ratio of non-phosphorylated PTEN (S380/T382/T383) was significantly elevated by 1.4-fold during late torpor compared with euthermic controls; this was coupled with an increase in substrate affinity for PIP3 (by 56%) in late torpor. Two proteolytic cleavage PEST motifs were identified in the C-terminus that overlapped with the phosphorylation sites of PTEN; pulse-proteolysis analysis of PTEN protein showed a decrease in protein stability during late torpor (Cm of urea decreased by 21%). Furthermore, the increase in PTEN activity observed was correlated with a decrease in PDK-1 phosphorylation by 32%, suggesting a downstream effect of PTEN activation during torpor. Transcriptional analysis showed that mRNA expression of pten and pdk-1 remain unchanged during hibernation, suggesting post-translation modification as the primary regulatory mechanism of PTEN function.Phosphorylation plays an important role in the regulation of PTEN enzymatic activity and protein stability.Activation of PTEN during torpor can regulate insulin signaling during periods of low energy state.
Keywords: Insulin signaling pathway; Ground squirrels; Hibernation; Protein phosphatase; Phosphorylation;

The glycosome is a unique organelle found in Kinetoplastids known to compartmentalize vital metabolic pathways including glycolysis, β-fatty acid oxidation and purine salvage. Organelle biogenesis depends on a network of proteins for trafficking and translocation of nascent protein into the glycosome. The interaction of the proteins LdPEX14 and LdPEX5 at the glycosome membrane is crucial for targeting proteins into this organelle.Deletion mutagenesis, pull-down, and bacterial two hybrid assay were used to map the LdPEX5 domain bound by LdPEX14. ELISA assays, ITC, intrinsic fluorescence and size exclusion chromatography to monitor binding and structural changes associated with the LdPEX5–LdPEX14 interaction.The LdPEX14 binding site was mapped to residues 280–300 on LdPEX5, a region containing the pentapeptide motif W293AQEY297. Deletion of this region abolished the LdPEX5–LdPEX14 interaction. Intrinsic fluorescence spectroscopy suggests that the stabilization of the LdPEX5–LdPEX14 complex is dependent on W293 docking into a hydrophobic pocket within the binding domain of ldpex14. Studies using a panel of synthetic peptides suggest a critical role for Y297 and to a lesser extent E296 in stabilizing the LdPEX5–LdPEX14 association.We show that the LdPEX14 binding site is more promiscuous and in contrast to other eukaryotic systems will accommodate a more degenerate pentapeptide motif with the sequences WXXXW or FXXXF, findings which may be exploited for potential drug design.
Keywords: Leishmania; Glycosome; Peroxin 14; Peroxin 5; Protein–protein interaction;

The effects of the cellular and infectious prion protein on the neuronal adaptor protein X11α by Jack O'Sullivan; Emma Comerford; Walid Rachidi; Michael Scott; Nigel M. Hooper; Hilary E.M. McMahon (2213-2221).
The neuronal adaptor protein X11α is a multidomain protein with a phosphotyrosine binding (PTB) domain, two PDZ (PSD_95, Drosophila disks-large, ZO-1) domains, a Munc Interacting (MI) domain and a CASK interacting region. Amongst its functions is a role in the regulation of the abnormal processing of the amyloid precursor protein (APP). It also regulates the activity of Cu/Zn Superoxide dismutase (SOD1) through binding with its chaperone the copper chaperone for SOD1. How X11α production is controlled has remained unclear.Using the neuroblastoma cell line, N2a, and knockdown studies, the effect of the cellular and infectious prion protein, PrPC and PrPSc, on X11α is examined.We show that X11α expression is directly proportional to the expression of PrPC, whereas its levels are reduced by PrPSc. We also show PrPSc to affect X11α at a functional level. One of the effects of prion infection is lowered cellular SOD1 levels, here by knockdown of X11α we identify that the effect of PrPSc on SOD1 can be reversed indicating that X11α is involved in prion disease pathogenesis.A role for the cellular and infectious prion protein, PrPC and PrPSc, respectively, in regulating X11α is identified in this work.Due to the multiple interacting partners of X11α, dysfunction or alteration in X11α will have a significant cellular effect. This work highlights the role of PrPC and PrPSc in the regulation of X11α, and provides a new target pathway to control X11α and its related functions.
Keywords: Prion; Scrapie; Neuronal adaptor protein; Superoxide dismutase;

13C NMR based profiling unveils different α-ketoglutarate pools involved into glutamate and lysine synthesis in the milk yeast Kluyveromyces lactis by D. Gorietti; E. Zanni; C. Palleschi; M. Delfini; D. Uccelletti; M. Saliola; C. Puccetti; A.P. Sobolev; L. Mannina; A. Miccheli (2222-2227).
The construction of efficient cell factories for the production of metabolites requires the rational improvement/engineering of the metabolism of microorganisms. The subject of this paper is directed towards the quantitative understanding of the respiratory/fermentative Kluyveromyces lactis yeast metabolism and its rag8 casein kinase mutant, taken as a model for all rag gene mutations. 13C NMR spectroscopy and [1,2-13C2]glucose were used as metabolic stable-isotope tracer to define the metabolic profiling of a K. lactis yeast and its derivative mutants. Rag8 showed a decrease of all 13C glutamate fractional enrichments, except for [4-13C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribution of a [4-13C]ketoglutarate pool not originating from mitochondria were suggested. 13C lysine enrichments confirmed the presence of two compartmentalized α-ketoglutarate (α-KG) pools participating to glutamate and lysine synthesis.Moreover, an increased transaldolase, as compared to transketolase activity, was observed in the rag8 mutant by 13C NMR isotopomer analysis of alanine. 13C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, 13C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized α-KG pool.A compartmentalization of the α-KG pools involved in lysine biosynthesis has been revealed for the first time in K. lactis. Given its great impact in metabolic engineering field, its existence should be validated/compared with other yeasts and/or fungal species.
Keywords: Yeasts; 13C NMR; Isotopomer analysis; Casein kinase; Lysine synthesis; Metabolic profiling;

Arginine-containing peptides as potent inhibitors of VIM-2 metallo-β-lactamase by Caitlyn M. Rotondo; Laura Marrone; Valerie J. Goodfellow; Ahmad Ghavami; Geneviève Labbé; James Spencer; Gary I. Dmitrienko; Stefan Siemann (2228-2238).
Metallo-β-lactamases (MBLs) play an important role in the emergence of microbial resistance to β-lactam antibiotics, and are hence considered targets for the design of novel therapeutics. We here report on the inhibitory effect of peptides containing multiple arginine residues on VIM-2, a clinically important MBL from Pseudomonas aeruginosa.Enzyme kinetic assays in combination with fluorescence spectroscopy and stopped-flow UV–Vis spectrophotometry were utilized to explore the structure–activity relationship of peptides as inhibitors of VIM-2.Our studies show that the inhibitory potency of the investigated peptides was mainly dependent on the number of arginine residues in the center of the peptide sequence, and on the composition of the N-terminus. The most potent inhibitors were found to curtail enzyme function in the mid-to-low nanomolar range. Salts generally reduced peptide-mediated inhibition. Analysis of the mode of inhibition suggests the peptides to act as mixed-type inhibitors with a higher affinity for the enzyme–substrate complex. Stopped-flow UV–Vis and fluorescence studies revealed the peptides to induce rapid protein aggregation, a phenomenon strongly correlated to the peptides' inhibitory potency. Inhibition of IMP-1 (another subclass B1 MBL) by the peptides was found to be much weaker than that observed with VIM-2, a finding which might be related to subtle molecular differences in the protein surfaces.The reported data indicate that arginine-containing peptides can serve as potent, aggregation-inducing inhibitors of VIM-2, and potentially of other MBLs.Arginine-containing peptides can be considered as a novel type of potent MBL inhibitors.Display Omitted
Keywords: Antibiotic resistance; Metallo-β-lactamases; Peptide inhibitors; Protein aggregation;

Protein N-homocysteinylation: From cellular toxicity to neurodegeneration by Gurumayum Suraj Sharma; Tarun Kumar; Tanveer Ali Dar; Laishram Rajendrakumar Singh (2239-2245).
Homocysteine (Hcy) is a sulfur containing non-protein amino acid that occupies a central role in metabolism of thiol compounds. The past decade had noticed an explosion in interests of Hcy and this very interest came primarily from the fact that increased Hcy level is related to various neurodegenerative and vascular complications.Several factors responsible for the Hcy-associated neurotoxicity have been proposed and well documented in literature, including oxidative stress and apoptosis. In addition, protein covalent modification by the metabolite of Hcy, Hcy thiolactone (HTL), has now been shown to be another cause of cellular Hcy toxicity. This mechanism, termed as “protein N-homocysteinylation”, is known to result in protein denaturation, enzyme inactivation and even amyloid formation. The role of protein N-homocysteinylation and the resulting consequences with regard to neurodegeneration have not yet been extensively discussed. The present review describes major advances in understanding protein N-homocysteinylation and their role in neurodegeneration.Formation of protein aggregates/amyloids are crucial events in various human pathologies including neurodegenerative diseases. Since elevated Hcy has been closely linked to neurodegeneration, N-homocysteinylation-induced protein modification and aggregates/amyloids formation could be one possible mechanism for the neurodegenerative conditions.The information highlighted here provides us an understanding of the role protein modification by N-homocysteinylation in neurodegenerative diseases.Display Omitted
Keywords: Amyloids; Homocystinuria; Homocysteine; Homocysteine thiolactone; Neurodegenerative disease; Protein N-homocysteinylation;

Bacterial appendage-dependent GH30 glucuronoxylan hydrolases recognize the substrate through an ionic interaction of a conserved positively charged arginine with the carboxyl group of 4-O-methyl-d-glucuronic acid. One of the options to verify this interaction is preparation of enzyme mutants. An alternative approach is a chemical modification of the substrate, glucuronoxylan, in which the free carboxyl group in all residues of MeGlcA is eliminated.In this work the carboxyl groups of 4-O-methyl-d-glucuronic acid residues of an alkali extracted beechwood xylan were esterified with methanol. A water-soluble fraction of the polysaccharide methyl ester was converted by NaBH4 reduction to the second soluble derivative, 4-O-methylglucoxylan. Specific activities of several endoxylanases (EXs) of GH families 10, 11 and 30 were determined on glucuronoxylan, and its two new uncharged derivatives.Elimination of the free carboxyl group from the polysaccharide did not influence activities of GH10 EXs, but resulted in 50% decrease of specific activity of GH11 EXs, and led to more than 300-fold reduction of specific activity of Erwinia chrysanthemi GH30 xylanase.These results confirm the crucial role of the interactions between GH30 xylanases and the MeGlcA carboxyl group for efficient cleavage of the polysaccharide. Analysis of the hydrolysis products by TLC and MS confirmed that all three types of xylanases hydrolyzed uncharged glucuronoxylans similarly as the original one.The uncharged glucuronoxylan derivatives will be useful to differentiate GH30 xylanases with various degree of selectivity for glucuronoxylan, including fungal enzymes without the conserved arginine.Display Omitted
Keywords: Glucuronoxylan; 4-O-methylglucuronic acid; Carboxyl group; 4-O-methylglucoxylan; Mode of action; Endoxylanase; Glycoside hydrolase families;

Prooxidant and antioxidant properties of salicylaldehyde isonicotinoyl hydrazone iron chelators in HepG2 cells by Andres A. Caro; Ava Commissariat; Caroline Dunn; Hyunjoo Kim; Salvador Lorente García; Allen Smith; Harrison Strang; Jake Stuppy; Linda P. Desrochers; Thomas E. Goodwin (2256-2264).
Salicylaldehyde isonicotinoyl hydrazone (SIH) is an iron chelator of the aroylhydrazone class that displays antioxidant or prooxidant effects in different mammalian cell lines. Because the liver is the major site of iron storage, elucidating the effect of SIH on hepatic oxidative metabolism is critical for designing effective hepatic antioxidant therapies.Hepatocyte-like HepG2 cells were exposed to SIH or to analogs showing greater stability, such as N′-[1-(2-Hydroxyphenyl)ethyliden]isonicotinoyl hydrazide (HAPI), or devoid of iron chelating properties, such as benzaldehyde isonicotinoyl hydrazone (BIH), and toxicity, oxidative stress and antioxidant (glutathione) metabolism were evaluated.Autoxidation of Fe2 + in vitro increased in the presence of SIH or HAPI (but not BIH), an effect partially blocked by Fe2 + chelation. Incubation of HepG2 cells with SIH or HAPI (but not BIH) was non-toxic and increased reactive oxygen species (ROS) levels, activated the transcription factor Nrf2, induced the catalytic subunit of γ-glutamate cysteine ligase (Gclc), and increased glutathione concentration. Fe2 + chelation decreased ROS and inhibited Nrf2 activation, and Nrf2 knock-down inhibited the induction of Gclc in the presence of HAPI. Inhibition of γ-glutamate cysteine ligase enzymatic activity inhibited the increase in glutathione caused by HAPI, and increased oxidative stress.SIH iron chelators display both prooxidant (increasing the autoxidation rate of Fe2 +) and antioxidant (activating Nrf2 signaling) effects.Activation by SIH iron chelators of a hormetic antioxidant response contributes to their antioxidant properties and modulates the anti- and pro-oxidant balance.
Keywords: Salicylaldehyde isonicotinoyl hydrazone; Iron; Oxidative stress; Antioxidant; Glutathione; Nrf2;

Dolichol phosphate mannose synthase from the pathogenic yeast Candida albicans is a multimeric enzyme by Mateusz Juchimiuk; Joanna Kruszewska; Grażyna Palamarczyk (2265-2275).
Dolichol phosphate mannose synthase (DPMS) is a key enzyme in N- and O-linked glycosylations and glycosylphosphatidylinositol (GPI)-anchor synthesis. DPMS generates DPM, the substrate for mentioned processes, by the transfer of mannosyl residue from GDP-Man to dolichol phosphate. Here we describe the role of DPMS for Candida albicans physiology with emphasis on the cell wall composition and morphogenesis. C. albicans genes for DPMS subunits were cloned, tagged and expressed in Saccharomyces cerevisiae. The C. albicans strains with controlled expression of DPM genes were constructed and analyzed. Gene expression and enzyme activities were measured using RT-PCR and radioactive substrate. Sensitivities against chemical agents were tested with microdilution method. The composition of the cell wall was estimated by HPLC. Glycosylation status of the marker protein was analyzed by Western blot. Morphological differentiation of the strains was checked on the media promoting hyphae and chlamydospore formation.We demonstrate that C. albicans DPMS consists of three interacting subunits, among which Dpm1 and Dpm3 are indispensable, whereas Dpm2 increases enzymatic activity. Lowered expression of DPMS genes results in decreased DPMS activity, increased susceptibility to cell wall perturbing agents and in altered cell wall composition. Mutants Tetp-DPM1 and Tetp-DPM3 show defective protein glycosylation and are impaired in hyphae and chlamydospore formation.DPMS from C. albicans, opposite to S. cerevisiae, belongs to the family of DPMS with multimeric protein structure.This work provides important data about factors required for a proper protein glycosylation and for morphogenesis of pathogenic yeast C. albicans.
Keywords: Candida albicans; Glycosylation; Dolichol phosphate mannose synthase; Cell wall;

Some reports indicate that NPs are ingested by cells via different mechanisms, including phagocytosis. In contrast, the direct role of NPs on the phagocytic process is not well documented. The aim of this study was to determine if titanium dioxide (TiO2), zinc oxide (ZnO) and cerium dioxide (CeO2) NPs, could alter the ability of neutrophils to exert phagocytosis.Freshly isolated human neutrophils were incubated with NPs and their ability to phagocytose opsonized sheep red blood cells (SRBCs) or fluorescent latex beads (LBs) was assessed by optical and fluorescence microscopy, respectively. Syk activation was assessed by western blot experiments and a pharmacological approach with piceatannol, a Syk inhibitor, was used to determine its role in NPs-induced neutrophils. The cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) was used as a positive control.All tested NPs enhanced the ability of neutrophil to phagocytose SRBCs and LBs. Syk was activated in NPs-induced neutrophils as evidenced by its increased tyrosine phosphorylation level vs controls and the ability of NPs-induced phagocytosis was reversed by piceatannol.We found that the tested NPs enhanced phagocytosis, although at different degree, and this occurred by a Syk-dependent mechanism.This is the first study demonstrating that NPs, by themselves, can directly enhance FcR-mediated (opsonized SRBCs) and complement-mediated (LBs) phagocytosis. Moreover, as part of their mode of action, we determined that NPs can act similarly to GM-CSF leading to Syk activation involved in phagocytosis. This has to be taken under consideration for future nanobiology and nanomedicine studies.
Keywords: Nanobiology; Nanoparticles; Neutrophils; Phagocytosis; Syk;

Mode of DNA binding with γ-butyrolactone receptor protein CprB from Streptomyces coelicolor revealed by site-specific fluorescence dynamics by Anwesha Biswas; Satya Narayan; Mamata V. Kallianpur; G. Krishnamoorthy; Ruchi Anand (2283-2292).
The γ-butyrolactone (GBL) binding transcription factors in Streptomyces species are known for their involvement in quorum sensing where they control the expression of various genes initiating secondary metabolic pathways. The structurally characterized member of this family CprB from Streptomyces coelicolor had earlier been demonstrated to bind a multitude of sequences containing a specific binding signature. Though structural breakthrough has been obtained for its complex with a consensus DNA sequence there is, however a dearth of information regarding the overall and site specific dynamics of protein–DNA interaction.To delineate the effect of CprB on the bound DNA, changes in motional dynamics of the fluorescent probe 2-aminopurine were monitored at three conserved base positions (5th, 12th and 23rd) for two DNA sequences: the consensus and the biologically relevant cognate element, on complex formation.The changes in lifetime and generalized order parameter revealed a similarity in the binding pattern of the protein to both sequences with greater dynamic restriction at the end positions, 5th and 23rd, as compared to the middle 12th position. Also differences within this pattern demonstrated the influence of even small changes in sequence on protein interactions.Here the study of motional dynamics was instrumental in establishing a structural footprint for the cognate DNA sequence and explaining the dynamics for the consensus DNA from structural correspondence.Motional dynamics can be a powerful tool to efficiently study the mode of DNA binding to proteins that interact differentially with a plethora of DNA sequences, even in the absence of structural breakthrough.Display Omitted
Keywords: 2-Aminopurine; γ-Butyrolactone; Anisotropy; Fluorescence lifetime;

H2S-induced S-sulfhydration of pyruvate carboxylase contributes to gluconeogenesis in liver cells by YoungJun Ju; Ashley Untereiner; Lingyun Wu; Guangdong Yang (2293-2303).
Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H2S) possesses diverse roles in the liver, affecting lipoprotein synthesis, insulin sensitivity, and mitochondrial biogenesis. H2S S-sulfhydration is now proposed as a major mechanism for H2S-mediated signaling. Pyruvate carboxylase (PC) is an important enzyme for gluconeogenesis. S-sulfhydration regulation of PC by H2S and its implication in gluconeogenesis in the liver have been unknown.Gene expressions were analyzed by real-time PCR and western blotting, and protein S-sulfhydration was assessed by both modified biotin switch assay and tag switch assay. Glucose production and PC activity was measured with coupled enzyme assays, respectively.Exogenously applied H2S stimulates PC activity and gluconeogenesis in both HepG2 cells and mouse primary liver cells. CSE overexpression enhanced but CSE knockout reduced PC activity and gluconeogenesis in liver cells, and blockage of PC activity abolished H2S-induced gluconeogenesis. H2S had no effect on the expressions of PC mRNA and protein, while H2S S-sulfhydrated PC in a dithiothreitol-sensitive way. PC S-sulfhydration was significantly strengthened by CSE overexpression but attenuated by CSE knockout, suggesting that H2S enhances glucose production through S-sulfhydrating PC. Mutation of cysteine 265 in human PC diminished H2S-induced PC S-sulfhydration and activity. In addition, high-fat diet feeding of mice decreased both CSE expression and PC S-sulfhydration in the liver, while glucose deprivation of HepG2 cells stimulated CSE expression.CSE/H2S pathway plays an important role in the regulation of glucose production through S-sulfhydrating PC in the liver.Tissue-specific regulation of CSE/H2S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes.
Keywords: H2S; CSE; PC; S-sulfhydration; Gluconeogenesis;

Exogenous control over intracellular acidification: Enhancement via proton caged compounds coupled to gold nanoparticles by Marilena Carbone; Gianfranco Sabbatella; Simonetta Antonaroli; Hynd Remita; Viviana Orlando; Stefano Biagioni; Alessandro Nucara (2304-2307).
The pH regulation has a fundamental role in several intracellular processes and its variation via exogenous compounds is a potential tool for intervening in the intracellular processes. Proton caged compounds (PPCs) release protons upon UV irradiation and may efficiently provoke intracellular on-command acidification. Here, we explore the intracellular pH variation, when purposely synthesized PCCs are coupled to gold nanoparticles (AuNPs) and dosed to HEK-293 cells. We detected the acidification process caused by the UV irradiation by monitoring the intensity of the asymmetric stretching mode of the CO2 molecule at 2343 cm− 1.The comparison between free and AuNPs functionalized proton caged compound demonstrates a highly enhanced CO2 yield, hence pH variation, in the latter case. Finally, PCC functionalized AuNPs were marked with a purposely synthesized fluorescent marker and dosed to HEK-293 cells. The corresponding fluorescence optical images show green grains throughout the whole cytoplasm.Display Omitted
Keywords: Proton caged compounds; Gold nanoparticles; pH monitoring; Intracellular acidification;

Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential by Maria Dimarogona; Efstratios Nikolaivits; Maria Kanelli; Paul Christakopoulos; Mats Sandgren; Evangelos Topakas (2308-2317).
Cutinases are serine hydrolases that degrade cutin, a polyester of fatty acids that is the main component of plant cuticle. These biocatalysts have recently attracted increased biotechnological interest due to their potential to modify and degrade polyethylene terephthalate (PET), as well as other synthetic polymers.A cutinase from the mesophilic fungus Fusarium oxysporum, named FoCut5a, was expressed either in the cytoplasm or periplasm of Escherichia coli BL21. Its X-ray structure was determined to 1.9 Å resolution using molecular replacement. The activity of the recombinant enzyme was tested on a variety of synthetic esters and polyester analogues.The highest production of recombinant FoCut5a was achieved using periplasmic expression at 16 °C. Its crystal structure is highly similar to previously determined Fusarium solani cutinase structure. However, a more detailed comparison of the surface properties and amino acid interactions revealed differences with potential impact on the biochemical properties of the two enzymes. FoCut5a showed maximum activity at 40 °C and pH 8.0, while it was active on three p-nitrophenyl synthetic esters of aliphatic acids (C2, C4, C12), with the highest catalytic efficiency for the hydrolysis of the butyl ester. The recombinant cutinase was also found capable of hydrolyzing PET model substrates and synthetic polymers.The present work is the first reported expression and crystal structure determination of a functional cutinase from the mesophilic fungus F. oxysporum with potential application in surface modification of PET synthetic polymers. FoCut5a could be used as a biocatalyst in industrial applications for the environmentally-friendly treatment of synthetic polymers.
Keywords: Heterologous expression; Escherichia coli; Serine esterase; PET modification; Crystal structure;

Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology by Mikhail E. Matlashov; Yulia A. Bogdanova; Galina V. Ermakova; Natalia M. Mishina; Yulia G. Ermakova; Evgeny S. Nikitin; Pavel M. Balaban; Shigeo Okabe; Sergey Lukyanov; Grigori Enikolopov; Andrey G. Zaraisky; Vsevolod V. Belousov (2318-2328).
SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use.Here we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer.Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate.SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo.The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.
Keywords: pH; Genetically encoded sensors; SypHer2; Neurons; Synapse; Regeneration; Imaging;

Modulation of aldose reductase activity by aldose hemiacetals by Francesco Balestri; Mario Cappiello; Roberta Moschini; Rossella Rotondo; Marco Abate; Antonella Del-Corso; Umberto Mura (2329-2339).
Glucose is considered as one of the main sources of cell damage related to aldose reductase (AR) action in hyperglycemic conditions and a worldwide effort is posed in searching for specific inhibitors of the enzyme. This AR substrate has often been reported as generating non-hyperbolic kinetics, mimicking a negative cooperative behavior. This feature was explained by the simultaneous action of two enzyme forms acting on the same substrate.The reduction of different aldoses and other classical AR substrates was studied using pure preparations of bovine lens and human recombinant AR.The apparent cooperative behavior of AR acting on glucose and other hexoses and pentoses, but not on tethroses, glyceraldehyde, 4-hydroxynonenal and 4-nitrobenzaldehyde, is generated by a partial nonclassical competitive inhibition exerted by the aldose hemiacetal on the reduction of the free aldehyde. A kinetic model is proposed and kinetic parameters are determined for the reduction of l-idose.Due to the unavoidable presence of the hemiacetal, glucose reduction by AR occurs under different conditions with respect to other relevant AR-substrates, such as alkanals and alkenals, coming from membrane lipid peroxidation. This may have implications in searching for AR inhibitors. The emerging kinetic parameters for the aldoses free aldehyde indicate the remarkable ability of the enzyme to interact and reduce highly hydrophilic and bulky substrates.The discovery of aldose reductase modulation by hemiacetals offers a new perspective in searching for aldose reductase inhibitors to be developed as drugs counteracting the onset of diabetic complications.Display Omitted
Keywords: Aldose reductase; Partial inhibition; Glucose; Hemiacetal; l-idose; Diabetes;

Functional characterization of zebrafish cytochrome P450 1 family proteins expressed in yeast by John J. Stegeman; Lars Behrendt; Bruce R. Woodin; Akira Kubota; Benjamin Lemaire; Denis Pompon; Jared V. Goldstone; Philippe Urban (2340-2352).
Zebrafish express five cytochrome P450 1 genes: CYP1A, CYP1B1, CYP1C1, CYP1C2, inducible by aryl hydrocarbon receptor agonists, and CYP1D1, a constitutively expressed CYP1A-like gene. We examined substrate selectivity of CYP1s expressed in yeast.CYP1s were expressed in W(R) yeast, engineered to over-express P450 reductase, via pYES/DEST52 and via pYeDP60. Microsomal fractions from transformed yeast were examined for activity with fluorogenic substrates, benzo[a]pyrene and testosterone. Modeling and docking approaches were used to further evaluate sites of oxidation on benzo[a]pyrene and testosterone.CYP1s expressed in yeast dealkylated ethoxy-, methoxy-, pentoxy- and benzoxy-resorufin (EROD, MROD, PROD, BROD). CYP1A and CYP1C2 had the highest rates of EROD activity, while PROD and BROD activities were low for all five CYP1s. The relative rates of resorufin dealkylation by CYP1C1, CYP1C2 and CYP1D1 expressed via pYeDP60 were highly similar to relative rates obtained with pYES/DEST52-expressed enzymes. CYP1C1 and CYP1C2 dealkylated substituted coumarins and ethoxy-fluorescein-ethylester, while CYP1D1 did not. The CYP1Cs and CYP1D1 co-expressed with epoxide hydrolase oxidized BaP with different rates and product profiles, and all three produced BaP-7,8,9,10-tetrol. The CYP1Cs but not CYP1D1 metabolized testosterone to 6β-OH-testosterone. However, CYP1D1 formed an unidentified testosterone metabolite better than the CYP1Cs. Testosterone and BaP docked to CYP homology models with poses consistent with differing product profiles.Yeast-expressed zebrafish CYP1s will be useful in determining further functionality with endogenous and xenobiotic compounds.Determining the roles of zebrafish CYP1s in physiology and toxicology depends on knowing the substrate selectivity of these enzymes.
Keywords: Zebrafish; Cytochrome P450 family 1; Recombinant CYP1 proteins; Yeast; Substrate selectivity;

During the maturation process, HIV capsid proteins self-assemble into polymorphic capsids. The strong polymorphism precludes high resolution structural characterization under in vivo conditions. In spite of the determination of structural models for various in vitro assemblies of HIV capsid proteins, the assembly mechanism is still not well-understood.We report 3D simulations of HIV capsid proteins by a novel coarse grain model that captures the backbone of the rigid segments in the protein accurately. The effects of protein dynamics on assembly are emulated by a static ensemble of subunits in conformations derived from molecular dynamics simulation.We show that HIV capsid proteins robustly assemble into hexameric lattices in a range of conditions where trimers of dimeric subunits are the dominant oligomeric intermediates. Variations of hexameric lattice curvatures are observed in simulations with subunits of variable inter-domain orientations mimicking the conformation distribution in solution. Simulations with subunits based on pentameric structural models lead to assembly of sharp curved structures resembling the tips of authentic HIV capsids, along a distinct pathway populated by tetramers and pentamers with the characteristic quasi-equivalency of viral capsids.Our results suggest that the polymorphism assembly is triggered by the inter-domain dynamics of HIV capsid proteins in solution. The assembly of highly curved structures arises from proteins in conformation with a highly specific inter-domain orientation.Our work proposes a mechanism of HIV capsid assembly based on available structural data, which can be readily verified. Our model can be applied to other large biomolecular assemblies.Display Omitted
Keywords: HIV; Capsid assembly; Coarse grain simulations;

Alcohol regulates the expression and function of protein kinase C epsilon (PKCε). In a previous study we identified an alcohol binding site in the C1B, one of the twin C1 subdomains of PKCε (Das et al., Biochem. J., 421, 405–13, 2009).In this study, we investigated alcohol binding in the entire C1 domain (combined C1A and C1B) of PKCε. Fluorescent phorbol ester, SAPD and fluorescent diacylglycerol (DAG) analog, dansyl-DAG were used to study the effect of ethanol, butanol, and octanol on the ligand binding using fluorescence resonance energy transfer (FRET). To identify alcohol binding site(s), PKCεC1 was photolabeled with 3-azibutanol and 3-azioctanol, and analyzed by mass spectrometry. The effects of alcohols and the azialcohols on PKCε were studied in NG108-15 cells.In the presence of alcohol, SAPD and dansyl-DAG showed different extent of FRET, indicating differential effects of alcohol on the C1A and C1B subdomains. Effects of alcohols and azialcohols on PKCε in NG108-15 cells were comparable. Azialcohols labeled Tyr-176 of C1A and Tyr-250 of C1B. Inspection of the model structure of PKCεC1 reveals that these residues are 40 Å apart from each other indicating that these residues form two different alcohol binding sites.The present results provide evidence for the presence of multiple alcohol-binding sites on PKCε and underscore the importance of targeting this PKC isoform in developing alcohol antagonists.Display Omitted
Keywords: Protein kinase Cε (PKCε); Alcohol-binding site; Alcoholism; Fluorescence resonance energy transfer (FRET); Mass spectrometry (MS); Photoaffinity;

Evidence against resveratrol as a viable therapy for the rescue of defective ΔF508 CFTR by Ying Jai; Kalpit Shah; Robert J. Bridges; Neil A. Bradbury (2377-2384).
Resveratrol, a natural phenolic compound, has been reported to rescue mutant ΔF508 CFTR in expression systems and primary epithelial cells. Although this implies a therapeutic benefit to patients with CF, investigations were performed using resveratrol concentrations greatly in excess of those achievable in plasma. We evaluated the efficacy of resveratrol as a CFTR corrector in relevant primary airway cells, using physiologically achievable resveratrol concentrations.Cells expressing wt or ΔF508 CFTR were exposed to chronic or acute resveratrol. CFTR mRNA and protein expression were monitored. The effects of resveratrol on primary ΔF508 human airway cells were evaluated by equivalent current analysis using modified Ussing chambers.Consistent with previously published data in heterologous expression systems, high doses of resveratrol increased CFTR expression; however physiologically relevant concentrations were without effect. In contrast to heterologous expression systems, resveratrol was unable to increase mutant CFTR channel activity in primary airway cells. Elevated amiloride-sensitive currents, indicative of sodium transport and characteristically elevated in CF airway cells, were also unaffected by resveratrol.High concentrations of resveratrol can increase CFTR mRNA and protein in some cell types. In addition, acute resveratrol exposure can stimulate CFTR mediated chloride secretion, probably by increasing cellular cAMP levels. Resveratrol at physiologically achievable levels yielded no benefit in primary ΔF508 airway cells, either in terms of amiloride-sensitive currents of CFTR currents.Taken together, our results do not support the use of resveratrol supplements as a therapy for patients with cystic fibrosis. It is possible that further modifications of the resveratrol backbone would yield a more efficacious compound.
Keywords: CFTR; Cystic fibrosis; Resveratrol; Human airway cell culture; Conductance;

Selenocysteine-independent suppression of UGA codons in the archaeon Methanococcus maripaludis by Deniz Seyhan; Nico Jehmlich; Martin von Bergen; Julia Fersch; Michael Rother (2385-2392).
Proteins containing selenocysteine (sec) are found in Bacteria, Eukarya, and Archaea. While selenium-dependence of methanogenesis from H2  + CO2 in the archaeon Methanococcus maripaludis JJ is compensated by induction of a set of cysteine-containing homologs, growth on formate is abrogated in the absence of sec due to the dependence of formate dehydrogenase (Fdh) on selenium. Despite this dependence, formate-dependent growth occurs after prolonged incubation of M. maripaludis mutants lacking sec.To study this phenomenon, a M. maripaludis strain with only one Fdh isoform and an FdhA selenoprotein C-terminally tagged for affinity enrichment was constructed. Factors required for sec synthesis were deleted in this strain and translation of UGA in fdhA was analyzed physiologically, enzymatically, immunologically, and via mass spectrometry. M. maripaludis JJ mutants lacking sec synthesis grew at least five times more slowly than the wild type on formate due to a 20–35-fold reduction of Fdh activity. The enzyme in the mutant strains lacked sec but was still produced as a full-length protein. Peptide mass spectrometry revealed that both cysteine (cys) and tryptophan (trp) were inserted at the UGA encoding sec without apparent mutations in tRNAcys or tRNAtrp, respectively.We demonstrate that M. maripaludis has the inherent capacity to translate UGA with cys and trp; other mechanisms to replace sec with cys in the absence of selenium could thereby be ruled out.This study exemplifies how an organism uses the inherent flexibility in its canonical protein synthesis machinery to recover some activity of an essential selenium-dependent enzyme in the absence of sec.
Keywords: Methanococcus maripaludis; Methanogenesis; Formate dehydrogenase; Selenocysteine; Stop codon; Suppression;