Biochmical et Biophysica Acta - General Subjects

Syndicate content ScienceDirect Publication: Biochimica et Biophysica Acta (BBA) - General Subjects
ScienceDirect RSS
Updated: 8 years 17 weeks ago

Production of 4-hydroxybutyrate from succinate semialdehyde in butyrate biosynthesis in Porphyromonas gingivalis

Wed, 10/28/2015 - 09:17
Publication date: December 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12

Author(s): Yasuo Yoshida, Mitsunari Sato, Keiji Nagano, Yoshiaki Hasegawa, Takashi Okamoto, Fuminobu Yoshimura

Background Despite evidence demonstrating the importance of butyrate-producing bacteria in host health and disease, the characterization of enzymes responsible for butyrate production has not been fully elucidated in the periodontopathogen, Porphyromonas gingivalis. Methods LC-MS/MS and colorimetric analyses were employed to enzymatically characterize recombinant PGN_0724 in P. gingivalis as a succinate semialdehyde reductase. The concentration of short chain fatty acids in the culture supernatant of the wild-type bacteria and a mutant strain lacking the PGN_0724 gene were quantified using GC-MS. Results Incubation of recombinant PGN_0724 with succinate semialdehyde and NADH resulted in the production of 4-hydroxybutyrate as well as consumption of succinate semialdehyde. Double reciprocal plots showed that the reaction catalyzed by the PGN_0724 protein was associated with a ternary complex mechanism. The growth speed and final turbidity of the mutant strain were much lower than those of the wild-type cells. The capacity of the mutant strain to produce butyrate, isobutyrate, and isovalerate was 30%, 15%, and 45%, respectively, of that of the wild-type strain, while the mutant strain produced approximately 3.9-fold more propionate than the wild type. Conclusions The pathway responsible for butyrate production is important for the growth of P. gingivalis and appears to be associated with production of the other short chain fatty acids. General significance The aim of this study was to delineate the mechanisms involved in the production of 4-hydroxybutyrate, which is an intermediate in the biosynthetic pathway for production of butyrate, which is a virulence factor in P. gingivalis.





Editorial Board

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11









Protein N-homocysteinylation: From cellular toxicity to neurodegeneration

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Gurumayum Suraj Sharma, Tarun Kumar, Tanveer Ali Dar, Laishram Rajendrakumar Singh

Background 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. Scope of review 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. Major conclusions 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. General significance The information highlighted here provides us an understanding of the role protein modification by N-homocysteinylation in neurodegenerative diseases.
Graphical abstract




Role of P2X7 receptor in Clostridium perfringens beta-toxin-mediated cellular injury

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Masahiro Nagahama, Soshi Seike, Hidenori Shirai, Teruhisa Takagishi, Keiko Kobayashi, Masaya Takehara, Jun Sakurai

Background 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. Methods 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. Results 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. Conclusions The results of this study indicate that the P2X7 receptor plays a role in beta-toxin-mediated cellular injury. General significance P2X7 receptor is a potential target for the treatment of C. perfringens type C infection.





Distinctive malfunctions of calmodulin mutations associated with heart RyR2-mediated arrhythmic disease

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): 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

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.





Using an in vitro model to study oxidised protein accumulation in ageing fibroblasts

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Nae Shiozawa-West, Rachael A. Dunlop, Kenneth J. Rodgers

Background 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. Methods 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. Results 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. Conclusions Our results highlight the importance of the HSP/chaperone system in oxidised protein metabolism, particularly in ageing cells. General significance 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.





Omega-3 DHA- and EPA–dopamine conjugates induce PPARγ-dependent breast cancer cell death through autophagy and apoptosis

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Daniela Rovito, Cinzia Giordano, Pierluigi Plastina, Ines Barone, Francesca De Amicis, Loredana Mauro, Pietro Rizza, Marilena Lanzino, Stefania Catalano, Daniela Bonofiglio, Sebastiano Andò

Background 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. Methods and results 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. Conclusions 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. General significance 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.





Post-translational regulation of PTEN catalytic function and protein stability in the hibernating 13-lined ground squirrel

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Cheng-Wei Wu, Ryan A. Bell, Kenneth B. Storey

Background 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. Methods 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. Results 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. Conclusion Phosphorylation plays an important role in the regulation of PTEN enzymatic activity and protein stability. General significance Activation of PTEN during torpor can regulate insulin signaling during periods of low energy state.





The Leishmania donovani peroxin 14 binding domain accommodates a high degeneracy in the pentapeptide motifs present on peroxin 5

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Hamed Hojjat, Armando Jardim

Background 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. Methods 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. Results and conclusions 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. General significance 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.





The effects of the cellular and infectious prion protein on the neuronal adaptor protein X11α

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Jack O'Sullivan, Emma Comerford, Walid Rachidi, Michael Scott, Nigel M. Hooper, Hilary E.M. McMahon

Background 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. Methods 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. Results 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. Conclusions A role for the cellular and infectious prion protein, PrPC and PrPSc, respectively, in regulating X11α is identified in this work. General significance 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.





13C NMR based profiling unveils different α-ketoglutarate pools involved into glutamate and lysine synthesis in the milk yeast Kluyveromyces lactis

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): D. Gorietti, E. Zanni, C. Palleschi, M. Delfini, D. Uccelletti, M. Saliola, C. Puccetti, A.P. Sobolev, L. Mannina, A. Miccheli

Background 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. Methods 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. Results 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. Conclusions 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. General significance 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.





Arginine-containing peptides as potent inhibitors of VIM-2 metallo-β-lactamase

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Caitlyn M. Rotondo, Laura Marrone, Valerie J. Goodfellow, Ahmad Ghavami, Geneviève Labbé, James Spencer, Gary I. Dmitrienko, Stefan Siemann

Background 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. Methods 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. Results 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. Conclusion The reported data indicate that arginine-containing peptides can serve as potent, aggregation-inducing inhibitors of VIM-2, and potentially of other MBLs. General significance Arginine-containing peptides can be considered as a novel type of potent MBL inhibitors.
Graphical abstract




The role of the glucuronoxylan carboxyl groups in the action of endoxylanases of three glycoside hydrolase families: A study with two substrate mutants

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): P. Biely, A. Malovíková, J. Hirsch, K.B.R. Morkeberg Krogh, A. Ebringerová

Background 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. Methods 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. Results 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. Conclusions 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. Significance 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.
Graphical abstract




Prooxidant and antioxidant properties of salicylaldehyde isonicotinoyl hydrazone iron chelators in HepG2 cells

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Andres A. Caro, Ava Commissariat, Caroline Dunn, Hyunjoo Kim, Salvador Lorente García, Allen Smith, Harrison Strang, Jake Stuppy, Linda P. Desrochers, Thomas E. Goodwin

Background 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. Methods 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. Results 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. Conclusions SIH iron chelators display both prooxidant (increasing the autoxidation rate of Fe2+) and antioxidant (activating Nrf2 signaling) effects. General significance Activation by SIH iron chelators of a hormetic antioxidant response contributes to their antioxidant properties and modulates the anti- and pro-oxidant balance.





Dolichol phosphate mannose synthase from the pathogenic yeast Candida albicans is a multimeric enzyme

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Mateusz Juchimiuk, Joanna Kruszewska, Grażyna Palamarczyk

Background 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. Methods 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. Results 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. Major conclusion DPMS from C. albicans, opposite to S. cerevisiae, belongs to the family of DPMS with multimeric protein structure. General significance This work provides important data about factors required for a proper protein glycosylation and for morphogenesis of pathogenic yeast C. albicans.





Nanoparticles enhance the ability of human neutrophils to exert phagocytosis by a Syk-dependent mechanism

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): K. Babin, D.M. Goncalves, D. Girard

Background 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. Methods 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. Results 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. Conclusions We found that the tested NPs enhanced phagocytosis, although at different degree, and this occurred by a Syk-dependent mechanism. General significance 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.





Mode of DNA binding with γ-butyrolactone receptor protein CprB from Streptomyces coelicolor revealed by site-specific fluorescence dynamics

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Anwesha Biswas, Satya Narayan, Mamata V. Kallianpur, G. Krishnamoorthy, Ruchi Anand

Background 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. Methods 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. Results 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. Conclusions 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. General significance 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.
Graphical abstract




H2S-induced S-sulfhydration of pyruvate carboxylase contributes to gluconeogenesis in liver cells

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): YoungJun Ju, Ashley Untereiner, Lingyun Wu, Guangdong Yang

Background 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. Methods 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. Results 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. Conclusions CSE/H2S pathway plays an important role in the regulation of glucose production through S-sulfhydrating PC in the liver. General significance Tissue-specific regulation of CSE/H2S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes.





Exogenous control over intracellular acidification: Enhancement via proton caged compounds coupled to gold nanoparticles

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Marilena Carbone, Gianfranco Sabbatella, Simonetta Antonaroli, Hynd Remita, Viviana Orlando, Stefano Biagioni, Alessandro Nucara

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 2343cm−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.
Graphical abstract




Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential

Wed, 10/28/2015 - 09:17
Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Maria Dimarogona, Efstratios Nikolaivits, Maria Kanelli, Paul Christakopoulos, Mats Sandgren, Evangelos Topakas

Background 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. Methods 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 1.9Å resolution using molecular replacement. The activity of the recombinant enzyme was tested on a variety of synthetic esters and polyester analogues. Results 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 pH8.0, 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. Conclusions 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. General significance FoCut5a could be used as a biocatalyst in industrial applications for the environmentally-friendly treatment of synthetic polymers.