http://www.biochemj.org Biochemical Journal RSS feed -- Immediate Publications 0264-6021 1470-8728 Biochemical Journal http://www.biochemj.org/images/BJ_Name.gif http://www.biochemj.org http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120618 R Tobe, M Yoo, N Fradejas, B A Carlson, S Calvo, V N Gladyshev, D L Hatfield 2012-05-17T12:46:10Z doi:10.1042/BJ20120618 Portland Press Limited 2012-05-17 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111920 K Hanzelka, L Skalniak, J Jura, S Lenzen, E Gurgul-Convey 2012-05-16T11:22:15Z doi:10.1042/BJ20111920 Portland Press Limited 2012-05-16 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120190 Candida albicans. The MDR1 protein with two transmembrane domains, each comprising six transmembrane helices, is interconnected with extracellular loops and intracellular loops (ICLs). The introduction of deletions and insertions through mutagenesis was used to address the role of the largest inter-domain ICL3 of the Mdr1 protein. Most of the progressive deletants, when overexpressed, eliminated the drug resistance. Notably, restoration of the length of the ICL3 by insertional mutagenesis did not restore the functionality of the protein. Interestingly, most of the insertion and deletant variants of ICL3 became amenable to trypsinization, yielding peptide fragments. The homology model of the Mdr1 protein showed that the molecular surface charge distribution was perturbed in most of the ICL3 mutant variants. Taken together, these results provide the first evidence that the central cytoplasmic loop (CCL) of the fungal MFS transporter of the DHA1 family is critical for the function of MDR. Unlike other homologous proteins, ICL3 has no apparent role in imparting substrate specificity or in the recruitment of the transporter protein.]]> A Mandal, A Kumar, A Singh, A M Lynn, K Kapoor, R Prasad 2012-05-16T11:13:53Z doi:10.1042/BJ20120190 Portland Press Limited 2012-05-16 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120533 D Munera, E Martinez, S Varyukhina, A Mahajan, J Ayala-Sanmartin, G Frankel 2012-05-16T10:58:34Z doi:10.1042/BJ20120533 Portland Press Limited 2012-05-16 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112169 K Kawauchi, W Tan, K Araki, F Abu Bakar, M Kim, H Fujita, H Hirata, Y Sawada 2012-05-15T16:08:19Z doi:10.1042/BJ20112169 Portland Press Limited 2012-05-15 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112086 Saccharomyces cerevisiae the Pho84 phosphate transporter acts as the main provider of phosphate to the cell using a proton symport mechanism, but also mediates rapid activation of the protein kinase A (PKA) pathway. These two features led to recognition of Pho84 as a transceptor. Although the physiological role of Pho84 has been studied in depth, the mechanisms underlying the transport- and sensor function are unclear. To obtain more insight into the structure-function relationships of Pho84, we have rationally designed and analyzed site-directed mutants. Using a three-dimensional model of Pho84 created on the basis of the GlpT permease, complemented with multiple-sequence alignments, we selected Arg-168 and Lys-492, and Asp-178, Asp-358 and Glu-473 as residues potentially involved in phosphate or proton binding, respectively, during transport. We found that Asp-358 (Helix 7) and Lys-492 (Helix 11) are critical for the transport function, and might be part of the putative substrate binding pocket of Pho84. Moreover, we show that alleles mutated in the putative proton-binding site Asp-358 are still capable of strongly activating PKA pathway targets despite their severely reduced transport activity. This indicates that signaling does not require transport and suggests that mutagenesis of amino acid residues involved in binding of the cotransported ion may constitute a promising general approach to separate the transport and signaling functions in transceptors.]]> D R. Samyn, L Ruiz-Pavon, M R. Andersson, Y Popova, J M Thevelein, B L. Persson 2012-05-15T16:05:31Z doi:10.1042/BJ20112086 Portland Press Limited 2012-05-15 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120248 2+ is thought to be central to the channel’s ability to regulate reactive oxygen species (ROS) production. However, channel-mediated entry of Mg²⁺ and Zn²⁺ have also been implicated in cell death. Here we show that depletion of TRPM7 by RNA interference in fibroblasts increases cell resistance to apoptotic stimuli by decreasing ROS levels in a Mg²⁺-dependent manner. Depletion of TRPM7 lowered cellular Mg²⁺, decreased the concentration of ROS and lessened p38 MAP kinase and JNK activation as well as decreased caspase-3 activation and PARP cleavage in response to apoptotic stimuli. Re-expression of TRPM7 or of a kinase-inactive mutant of TRPM7 in TRPM7-knockdown cells increased cellular Mg²⁺ and ROS levels, as did expression of the Mg²⁺ transporter SLC41A2. In addition, expression of SLC41A2 increased TRPM7-knockdown cells’ sensitivity to apoptotic stimuli as well as boosted ROS generation in response to cell stress. Together these data uncover an essential role for Mg²⁺ in TRPM7’s control of cell survival and in the regulation of cellular ROS levels.]]> H Chen, L Su, O González-Pagán, J D. Overton, L W. Runnels 2012-05-15T15:52:01Z doi:10.1042/BJ20120248 Portland Press Limited 2012-05-15 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120406 M A Rieger, T Duellman, C Hooper, M Ameka, J C Bakowska, B D Cuevas 2012-05-14T13:25:38Z doi:10.1042/BJ20120406 Portland Press Limited 2012-05-14 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111956 MS analysis of purified histones, revealing previously unseen patterns of histone acetylation in Arabidopsis. Immunoblot analysis revealed an increase in relative abundance of the H3 acetylated N-terminus, and a global decrease in hyperacetylation of H4 in response to DNA damage induced by X-rays. Conversely, mutants in the key DNA damage signalling factor ATAXIA TELANGIECTASIA MUTATED (ATM) display increased histone acetylation upon irradiation, linking the DNA damage response with dynamic changes in histone modification in plants.]]> G E Drury, A A Dowle, D A Ashford, W M Waterworth, J Thomas, C E West 2012-05-11T10:42:02Z doi:10.1042/BJ20111956 Portland Press Limited 2012-05-11 BJ Plant http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111923 N-linked glycosylation is a critical determinant of protein structure and function, regulating processes such as protein folding, stability, localization, ligand-receptor binding and intracellular signaling. The type II TGF-b receptor (TbRII) plays a crucial role in the TGF-b signaling pathway. Although N-linked glycosylation of TbRII was first demonstrated over a decade ago, it was unclear how this modification influenced TbRII biology. Here, we show that inhibiting N-linked glycosylation process successfully hinders binding of TGF-b1 to TbRII and subsequently renders cells resistant to TGF-b signaling. Lung cancer cell line A549, gastric carcinoma cell line MKN1 and immortal cell line HEK293 exhibit reduced TGF-b signaling when either treated with two inhibitors, including tunicamycin (a potent N-linked glycosylation inhibitor) and kifunensine (an inhibitor of ER and Golgi mannosidase I family members), or introduced with the non-glycosylated mutant version of TbRII. We demonstrate that defective N-linked glycosylation prevents TbRII proteins from being transported to the cell surface. Moreover, we clearly show that not only complex type but also high-mannose type of TbRII can be localized on the cell surface. Collectively, these findings demonstrate that N-linked glycosylation is essentially required for the successful cell surface transportation of TbRII, suggesting a novel mechanism, which the TGF-b sensitivity can be regulated by N-linked glycosylation levels of TbRII.]]> Y Kim, J Park, H Lee, S Lee, S Kim 2012-05-10T10:37:40Z doi:10.1042/BJ20111923 Portland Press Limited 2012-05-10 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120504 B W Cook, G S Shaw 2012-05-08T13:05:23Z doi:10.1042/BJ20120504 Portland Press Limited 2012-05-08 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111575 Q Lin, J Wang, C Childress, W Yang 2012-05-03T15:38:26Z doi:10.1042/BJ20111575 Portland Press Limited 2012-05-03 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120169 Helicobacter pylori. Uptake in the latter is mediated by UreI, a Urea Amide Channel family member. Here we describe the structure and function of UAC_Bc, a homologue from Bacillus cereus. The purified channel was found to be permeable not only to urea but also to other small amides. Circular dichroism and infrared spectroscopy revealed a structure comprising mainly α-helices, oriented approximately perpendicular to the membrane. Consistent with this finding, site-directed fluorescent labelling indicated the presence of 7 transmembrane (TM) helices, with a cytoplasmic C-terminus. In detergent UAC_Bc exists largely as a hexamer, as demonstrated both by cross-linking and size exclusion chromatography. A 9 Å resolution projection map obtained by electron cryomicroscopy of 2-D crystals shows that the six protomers are arranged in a planar hexameric ring. Each exhibits six density features attributable to TM helices, surrounding a putative central channel, while an additional helix is peripherally located. Bioinformatic analyses allowed individual TM regions to be tentatively assigned to the density features, the resultant model enabling identification of residues likely to contribute to channel function.]]> G H.M. Huysmans, N Chan, J M. Baldwin, V L.G. Postis, S B. Tzokov, S E. Deacon, S Y.M. Yao, J D. Young, M J. McPherson, P A. Bullough, S A. Baldwin 2012-05-03T13:30:54Z doi:10.1042/BJ20120169 Portland Press Limited 2012-05-03 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120439 vitro. In this article we analyze the data showing the dependence of this parameter upon muscle contractile state. Most remarkable is the effect of recently described calcium- independent contraction of permeabilized muscle fibers induced by elevated temperatures (30-37°C). We show that very similar strong spontaneous calcium-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibers that resulting in a disorganization of mitochondrial arrangement leading to significant increase of affinity for ADP. These data show that calcium-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore, use of their inhibitors is strongly advised at the step of permeabilization with careful washing of fibers or cells afterwards. Possible physiologically relevant relationship between calcium-regulated, ATP-dependent contraction and mitochondrial functional parameters is also discussed.]]> A V. Kuznetsov, R Guzun, F Boucher, R Bagur, T Kaambre, V A Saks 2012-05-03T10:36:08Z doi:10.1042/BJ20120439 Portland Press Limited 2012-05-03 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120038 O-acetylserine(thiol)lyase (OAS-TL), which replaces the activated acetyl group of O-acetylserine with sulfide. The enzyme is present in cytosol, plastids and mitochondria of plant cells. Solely the knockout of mitochondrial OAS-TL activity (oastlC) leads to significant reduction of growth in Arabidopsis thaliana. The reason for this phenotype is still enigmatic, since mitochondrial OAS-TL accounts only for approximately 5 % of total OAS-TL activity. In this study we demonstrate that sulfide specifically intoxicates complex IV activity but not electron transport through complex II + III in isolated mitochondria of oastlC plants. Loss of mitochondrial OAS-TL activity resulted in significant inhibition of dark respiration under certain developmental conditions. The abundance of mitochondrial encoded proteins and iron-sulfur cluster containing proteins was not affected in oastlC. Furthermore, oastlC seedlings were insensitive to cyanide, which is detoxified by b-cyano-alanine synthase in mitochondria at the expense of cysteine. These results indicate that in-situ biosynthesis of cysteine in mitochondria is not mandatory for translation, Fe-S cluster assembly and cyanide detoxification. Finally, we uncover an OAS-TL-independent detoxification system for sulfide in mitochondria of Arabidopsis that allows oastlC plants to cope with high sulfide levels caused by abiotic stresses.]]> H Birke, F H. Haas, L J. De Kok, J Balk, M Wirtz, R Hell 2012-05-02T13:23:44Z doi:10.1042/BJ20120038 Portland Press Limited 2012-05-02 BJ Plant http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120025 Cit9; anti-CE1), (2) citrullination of arginine at position 9 in ENO1 and NSE (ENO1^Cit9/NSE^Cit9; anti-CE1/2), and (3) citrullination of arginine at position 429 of NSE (NSE^Cit429; anti-CE2). Regardless of the total protein expression level, the levels of ENO1^Cit9 and NSE^Cit429 were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in the frontal cortex of patients with sporadic Creutzfeldt-Jakob disease and Alzheimer’s disease compared to controls. In a time- and dose-dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by calcium-dependent calpain-1, and leupeptin (calpain inhibitor I) abrogated this degradation. Surprisingly, using an affinity assay, the citrullination of enolase enhanced its plasminogen binding affinity, which was blocked by the lysine analog ε-aminocaproic acid. These findings suggest that PAD-mediated citrullination regulates the diverse physiological activities of enolase and that citrullinated enolase may be a candidate diagnostic/prognostic factor for degenerative diseases.]]> B Jang, Y Jeon, J Choi, M Park, J Kim, A Ishigami, N Maruyama, R I Carp, Y KIM, E Choi 2012-05-02T13:18:48Z doi:10.1042/BJ20120025 Portland Press Limited 2012-05-02 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120232 N-glycosylation has been linked to increased basal cell surface levels while N-glycosylation-deficient GLUT4 was found to be quickly degraded. As recycling and degradation of GLUT4 are positively correlated, we hypothesized that incorrect N-glycosylation of GLUT4 might reduce its intracellular retention resulting in an increased cell surface recycling, in increased basal cell surface levels, and in enhanced GLUT4 degradation. Here, we have studied N-glycosylation-deficient GLUT4 in detail in 3T3-L1 preadipocytes, 3T3-L1 adipocytes, and L6 myoblasts. We have found no alterations in retention, insulin response, internalization, or glucose transport activity. Degradation of the mutant molecule was increased, though once present at the cell surface, its degradation was identical to that of wild-type GLUT4. Our findings indicate that N-glycosylation is important for efficient trafficking of GLUT4 to its proper compartments, but once the transporter has arrived there, N-glycosylation plays no further major role in its intracellular traffic, nor in its functional activity.]]> N Zaarour, M Berenguer, Y Le Marchand-Brustel, R Govers 2012-04-30T14:17:26Z doi:10.1042/BJ20120232 Portland Press Limited 2012-04-30 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120528 Legionella pneumophila is an opportunistic pathogen and the causative agent of Legionnaires’ disease. Despite being exposed to many chemical compounds in its natural and man-made habitats (natural aquatic biotopes and man-made water systems), L. pneumophila is able to adapt and survive in these environments. The molecular mechanisms by which this bacterium detoxifies these chemicals remain poorly understood. In particular, the expression and functions of xenobiotic-metabolizing enzymes (XME) that could contribute to chemical detoxification in L. pneumophila have been poorly documented at the molecular and functional levels. We report here the identification and biochemical and functional characterization of a unique acetyltransferase that metabolizes aromatic amine chemicals in three characterized clinical strains of L. pneumophila (Paris, Lens, and Philadelphia). Strain-specific sequence variations in this enzyme, an atypical member of the arylamine N-acetyltransferase family (EC 2.3.1.5), produce enzymatic variants with different structural and catalytic properties. Functional inactivation and complementation experiments showed that this acetyltransferase allows L. pneumophila to detoxify aromatic amine chemicals and grow in their presence. Our study provides a new enzymatic mechanism by which the opportunistic pathogen L. pneumophila biotransforms and detoxifies toxic aromatic chemicals. These data also emphasize the role of xenobiotic-metabolizing enzymes in the environmental adaptation of certain prokaryotes.]]> X Kubiak, D Dervins-Ravault, B Pluvinage, A Chaffotte, L Gomez-Valero, J Dairou, F Busi, J Dupret, C Buchrieser, F Rodrigues-Lima 2012-04-30T13:36:25Z doi:10.1042/BJ20120528 Portland Press Limited 2012-04-30 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120136 W Zhang, D Dourado, P Alexandrino Fernandes, M João Ramos, B Mannervik 2012-04-26T11:21:40Z doi:10.1042/BJ20120136 Portland Press Limited 2012-04-26 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120173 2 allosteric site are formed by opposing N- and C-termini of subunits oriented antiparallel in a dimer. On the contrary, we show here that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in phosphofructokinase-deficient yeast restored growth on glucose. However, the N-terminus alone was not stable in vitro. The C-terminus is not catalytic but is needed for stability of the enzyme, as is the connecting peptide that normally joins the two domains (here included in the N-terminus). Co-expression of homologous, but not heterologous, N- and C-termini yielded stable, fully active enzymes in vitro with sizes and kinetic properties similar to those of the wild type tetrameric enzymes. This indicates that the separately translated domains can fold sufficiently well to bind to each other, that such binding of complementary domains is stable and that the alignment is sufficiently accurate and tight as to preserve metabolite binding sites and allosteric interactions.]]> O H. Martínez-Costa, V Sánchez, A Lázaro, E D. Hernández, K Tornheim, J J. Aragón 2012-04-25T09:49:43Z doi:10.1042/BJ20120173 Portland Press Limited 2012-04-25 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112166 Candida albicans RCH1 encodes a protein of 10-transmembrane domains, homologous to human SLC10A7, and localizes in the plasma membrane. Deletion of RCH1 confers hypersensitivity to high concentrations of extracellular calcium and tolerance to azoles and lithium, which phenocopies the deletion of CaPMC1 encoding the vacuolar calcium pump. Additive to CaPMC1 mutation, lack of RCH1 alone shows an increase in calcium sensitivity, calcium uptake and cytosolic calcium level. The calcium hypersensitivity is abolished by cyclosporin A and magnesium. In addition, deletion of RCH1 elevates the expression of CaUTR2, a downstream target of the calcium/calcineurin signaling. Mutational and functional analysis indicates that the Rch1p TM8 domain, but not the TM9 and TM10 domains, are required for its protein stability, cellular functions and subcellular localization. Therefore, Rch1p is a novel regulator of cytosolic calcium homeostasis, which expands the functional spectrum of the vertebrate SLC10 family.]]> L Jiang, J Alber, J Wang, W Du, X Yang, X Li, D Sanglard, J Geyer 2012-04-24T14:38:10Z doi:10.1042/BJ20112166 Portland Press Limited 2012-04-24 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112142 succination of proteins, increases in adipocytes grown in high glucose medium and in adipose tissues of type 2 diabetic mice. However, the metabolic mechanisms leading to increased fumarate and succination of protein in the adipocyte are unknown. Treatment of 3T3 cells with high glucose (30 mM vs. 5 mM) caused a significant increase in cellular ATP/ADP, NADH/NAD⁺, and the mitochondrial membrane potential. There was also a significant increase in cellular fumarate concentration and succination of proteins, which may be attributed to the increase in NADH/NAD⁺ and subsequent inhibition of Krebs cycle NAD⁺-dependent dehydrogenases. Chemical uncouplers, which dissipated the mitochondrial membrane potential and reduced the NADH/NAD­⁺ ratio, also decreased fumarate concentration and protein succination. High glucose plus metformin, an inhibitor of Complex I in the electron transport chain, caused an increase in fumarate and succination of protein. Thus, excess fuel supply (glucotoxicity) appears to create a pseudohypoxic environment (high NADH/NAD⁺ without hypoxia), which drives the increase in succination of protein. We propose that increased succination of proteins is an early marker of glucotoxicity and mitochondrial stress in adipose tissue in diabetes.]]> N Frizzell, S A Thomas, J A Carson, J W Baynes 2012-04-24T10:00:12Z doi:10.1042/BJ20112142 Portland Press Limited 2012-04-24 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112102 C. elegans belonging to the saposin-like protein superfamily and considered important elements of the nematode´s intestinal immune system. Here, we demonstrate that unlike the other members of the multifarious gene family (spps) coding for caenopores, spp-12 is expressed exclusively in two pharyngeal neurons. Recombinantly expressed SPP-12 binds to phospholipid membranes and forms pores in a pH-dependent manner characteristic for caenopores. Moreover, SPP-12 kills viable gram-positive bacteria, yeast cells and amoebae by permeabilizing their membranes suggesting a wide target cell spectrum. A spp-12 knock-out mutant is more susceptible to pathogenic Bacillus thuringiensis than wild-type worms and is tolerant to non-pathogenic bacteria. By contrast, SPP-1, a caenopore which gene is expressed in the intestine only and reported to be regulated by the same pathway as spp-12, is apparently non-protective against pathogenic B. thuringiensis, although displaying antimicrobial activity as well. The transcription of spp-1 is down-regulated in wild-type worms in the presence of pathogenic B. thuringiensis and a spp-1 knock-out mutant is hyposusceptible to this bacterium. This implies that SPP-12 but not SPP-1 contributes to resistance against B. thuringiensis, a natural pathogen of the nematode.]]> A Hoeckendorf, M Stanisak, M Leippe 2012-04-23T10:23:31Z doi:10.1042/BJ20112102 Portland Press Limited 2012-04-23 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120053 WT and SP-1 mutants, T578A, S586A and S587A. Expression of WT and mutant SP-1 increased MMP-9 promoter activity in alveolar macrophages. However, constitutively active Rac1 suppressed MMP-9 promoter activity in cells expressing SP-1_WT, SP-1_T578A, and SP-1_S587A, but not SP-1_S586A. Furthermore, constitutive ERK activation, which was inhibited by Rac1, significantly increased MMP-9 transcription in cells expressing SP-1_WT but not SP-1_S586A. Because Rac1 activation and ERK inactivation increased degradation of SP-1_WT and not SP-1_S586A, our results suggest that SP-1 stability mediated at S586 regulates MMP-9 transcription. Ex vivo, alveolar macrophages obtained from asbestosis patients had less MMP-9 expression that was associated with decreased SP-1 expression and ERK activation. These observations demonstrate that S586 in the PEST domain of SP-1 is important for MMP-9 gene expression in alveolar macrophages and highlight the importance of these proteins in pulmonary fibrosis.]]> S Murthy, A J Ryan, A Brent Carter 2012-04-23T10:09:55Z doi:10.1042/BJ20120053 Portland Press Limited 2012-04-23 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112113 Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. Here we show that purified recombinant OLEI01171 exhibites high esterase activity against the model esterase substrate α-naphthyl acetate at 5 °C – 30 °C with maximal activity at 15 °C – 20 °C. The esterase activity of OLEI01171 was stimulated 3-8 times by the addition of chloride or several other anions (0.1 M – 1.0 M). Compared to mesophilic PF00756 esterases, OLEI01171 exhibited lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 Å resolution and revealed a classical Ser hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to coordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. Our data suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by Lys had no significant effect on the OLEI01171 activity or thermostability, but resulted in a detectable increase of activity at 35 °C to 45 °C. Our work has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.]]> S Lemak, A Tchigvintsev, P Petit, R Flick, A U. Singer, G Brown, E Evdokimova, O Egorova, C Gonzalez, T N. Chernikova, M M. Yakimov, M Kube, R Reinhardt, P N. Golyshin, A Savchenko, A F. Yakunin 2012-04-23T09:00:00Z doi:10.1042/BJ20112113 Portland Press Limited 2012-04-23 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111588 E M Ribe, Y Y Jean, R L Goldstein, C Manzl, L Stefanis, A Villunger, C M. Troy 2012-04-19T15:17:20Z doi:10.1042/BJ20111588 Portland Press Limited 2012-04-19 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120448 1-micro­globulin (a₁m), an abundant protein in human blood plasma, which reveals the beta-barrel fold typical for lipocalins with a deep pocket lined by four loops at its open rim. Loop #1 harbours the residue Cys34 that is responsible for covalent cross-linking with plasma IgA. A single disulfide bond between Cys72 and Cys169 connects the C-terminal segment to the beta-barrel as in many other lipocalins. The exposed imidazole side chains of His122 and His123 in loop #4 give rise to a double Ni²⁺-binding site together with a crystallographic neighbour. The closest structural relatives of a₁m are the complement protein component C8gamma, the L-prostaglandin D synthase, and lipocalin 15, three other structurally characterized members of the lipocalin family in humans that have only distant sequence similarity. In contrast with these, a₁m is initially expressed as a bifunctional fusion protein with the protease inhibitor bikunin. Neither the electron density nor ESI-MS provide evidence for a chromophore bound to the recombinant a₁m, also known as "yellow-brown lipocalin". However, the three side chains of Lys92, Lys118, and Lys130 that were reported to be involved in covalent chromophore binding appear to be freely accessible to ligands accommodated in the hydrophobic pocket. A structural feature similar to the well known CP haem-binding motif indicates the presence of a haem-binding site within the loop region of a₁m, which explains previous biochemical findings and supports a physiological role in haem scavenging as well as redox-mediated detoxification]]> W Meining, A Skerra 2012-04-19T11:24:06Z doi:10.1042/BJ20120448 Portland Press Limited 2012-04-19 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111978 Symplocarpus renifolius and Arum maculatum are known to produce significant heat during the course of their floral development but using different regulatory mechanisms i.e., homoeothermic versus transient thermogenesis. To further clarify the molecular basis of species-specific thermogenesis in plants, we have here analyzed the native structures and expression patterns of the mitochondrial respiratory components in S. renifolius and A. maculatum. Our comparative analysis using blue native polyacrylamide gel electrophoresis combined with nano LC-MS/MS has revealed that the constituents of the respiratory complexes in both plants were basically similar, but that several mitochondrial components appeared to be differently expressed in their thermogenic organs. Namely, complex II in S. renifolius was detected as a 340 kDa product, suggesting an oligomeric or supramolecular structure in vivo. Moreover, the expression of an external NAD(P)H dehydrogenase was found to be higher in A. maculatum than in S. renifolius whereas an internal NAD(P)H dehydrogenase was expressed to a similar level in both species. Alternative oxidase was detected as smear-like signals that were elongated on the first dimension with a peak at around 200 kDa in both species. The significance and implication of these data are discussed in terms of thermoregulation in plants.]]> Y Kakizaki, A L. Moore, K Ito 2012-04-19T09:46:25Z doi:10.1042/BJ20111978 Portland Press Limited 2012-04-19 BJ Plant http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111984 Neisseria species N. meningitidis and N. gonorrhoeae are able to respire in the absence of oxygen, using nitrite as an alternative electron acceptor. Nitrite reductase (encoded by aniA) is tightly regulated by four transcriptional regulators: FNR, NarP, FUR and NsrR. The four regulators control expression of aniA in N. meningitidis by binding to specific and distinct regions of the promoter. We show here that FUR and NarP are both required for induction of expression of aniA in N. meningitidis, and that they bind adjacent to one another in a non-cooperative manner. Activation via FUR/NarP is dependent on their topological arrangement relative to the RNA polymerase binding site. Analysis of the sequence of the aniA promoters from multiple N. meningitidis and N. gonorrhoeae strains indicates that there are species-specific single nucleotide polymorphisms, in regions predicted to be important for regulator binding. These sequence differences alter both the in vitro DNA binding and the promoter activation in intact cells by key activators FNR (oxygen sensor) and NarP (which is activated by nitrite in N. meningitidis). The weak relative binding of FNR to the N. gonorrhoeae aniA promoter (compared to N. meningitidis) is compensated for by a higher affinity of the gonococcal aniA promoter for NarP. Despite containing nearly identical genes for catalysing and regulating denitrification, variations in the promoter for the aniA gene appear to have been selected to enable the two pathogens to tune differentially their responses to environmental variables during the aerobic-anaerobic switch.]]> J Edwards, D Quinn, K Rowbottom, J L Whittingham, M J. Thomson, J W. B. Moir 2012-04-18T11:25:28Z doi:10.1042/BJ20111984 Portland Press Limited 2012-04-18 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120433 K Yamamoto, A Trad, A Baumgart, L Hüske, I Lorenzen, A Chalaris, J Grötzinger, T Dechow, J Scheller, S Rose-John 2012-04-17T15:27:08Z doi:10.1042/BJ20120433 Portland Press Limited 2012-04-17 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111704 Zea mays ZmPIP1s, ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.]]> G P Bienert, D Cavez, A Besserer, M C Berny, D Gilis, M Rooman, F Chaumont 2012-04-17T11:29:57Z doi:10.1042/BJ20111704 Portland Press Limited 2012-04-17 BJ Plant http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120471 2+-handling proteins such as IP₃ receptors and TRPC channels. Here we present evidence for a role of Homer proteins in the STIM1/Orai1 association, as well as in the TRPC1/IP₃RII interaction, which might be of relevance in platelet function. Treatment of human platelets with thapsigargin or thrombin, results in Ca²⁺-independent association of Homer1 with TRPC1 and IP₃RII. In addition, thapsigargin and thrombin enhanced association of Homer1 with STIM1 and Orai1 in a Ca²⁺-dependent manner. Interference with Homer function by introduction into cells of the synthetic PPKKFR peptide, which emulates the proline-rich sequences of the PPXXF motif, reduced both STIM1/Orai1 and TRPC1/ IP₃RII associations, as compared to the introduction of the inactive PPKKRR peptide. The PPKKFR peptide attenuates thrombin-evoked Ca²⁺ entry and the maintenance of thapsigargin-induced store-operated Ca²⁺ entry. Finally, the PPKKFR peptide attenuated thrombin-induced platelet aggregation. These findings support an important role for Homer proteins in thrombin-stimulated platelet function, which is likely mediated by the support of agonist-induced Ca²⁺ entry.]]> I JARDIN, L ALBARRAN, N BERMEJO, G M Salido, J A. Rosado 2012-04-17T10:53:28Z doi:10.1042/BJ20120471 Portland Press Limited 2012-04-17 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120499 Z Zheng, S Itri Amran, J Zhu, O Schmidt-Kittler, K W Kinzler, B Vogelstein, P R Shepherd, P E Thompson, I G Jennings 2012-04-13T13:47:54Z doi:10.1042/BJ20120499 Portland Press Limited 2012-04-13 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111840 O Braten, N Shabek, Y Kravtsova-Ivantsiv, A Ciechanover 2012-04-13T11:23:37Z doi:10.1042/BJ20111840 Portland Press Limited 2012-04-13 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120304 Xenopus laevis oocytes and show by mutation that phosphorylation at a four residue cluster is required for oocyte maturation. We further demonstrate that these phosphorylations are critical for cytoplasmic polyadenylation but not for ePAB’s inherent ability to promote translation. Our data provide the first insight into the role of post-translational modifications in regulating PABP protein activity in vivo.]]> K Friend, M Brook, F Bezirci, M D Sheets, N K Gray, E Seli 2012-04-13T10:52:47Z doi:10.1042/BJ20120304 Portland Press Limited 2012-04-13 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112103 trans double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the head group and access to the amide N-H group of ceramide is indispensible for Bax binding indicating that Bax may be interacting with the polar portion of the ceramide channel facing the bulk phase. On the contrary, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the head group. This study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis while short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL including 2-methoxyantimycin A₃, ABT-737, and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.]]> M N Perera, S H Lin, Y K Peterson, A Bielawska, Z M. Szulc, R Bittman, M Colombini 2012-04-11T13:31:33Z doi:10.1042/BJ20112103 Portland Press Limited 2012-04-11 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120408 I Lamberto, H Qin, R Noberini, L Premkumar, C Bourgin, S Riedl, J Song, E B. Pasquale 2012-04-10T14:53:24Z doi:10.1042/BJ20120408 Portland Press Limited 2012-04-10 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112119 Y Shifrin, V I Pinto, A Hassanali, P D Arora, C A McCulloch 2012-04-10T14:28:11Z doi:10.1042/BJ20112119 Portland Press Limited 2012-04-10 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111383 2-GPI (β ₂-glycoprotein I) is a plasma glycoprotein ascribed with an anti-angiogenic function; however, the biological role and molecular basis of its action in cell migration remain unknown. The aim of this study was to assess the contribution of β ₂-GPI to HAEC (human aortic endothelial cell) migration and the details of its underlying mechanism. Using wound healing and Boyden chamber assays, we found that β ₂-GPI inhibited endothelial cell migration, which was restored by the neutralizing antibody. NF-кB (nuclear factor-кB) inhibitors and lentiviral siRNA (small interfering RNA) silencing of NF-кB significantly attenuated the inhibitory effect of β ₂-GPI on cell migration. Moreover, β ₂-GPI was found to induce IκBa (inhibitor of NF-кB) phosphorylation, and translocation of p65 and p50. We further demonstrated that mRNA and protein levels of eNOS (endothelial nitric oxide synthase) and NO (nitric oxide) production were all increased by β₂-GPI and these effects were remarkably inhibited by NF-кB inhibitors and siRNAs of p65 and p50. Furthermore, β₂-GPI-mediated inhibition of cell migration was reversed by eNOS inhibitors and eNOS siRNAs. Our findings provide novel insight into the ability of β₂-GPI to inhibit endothelial cell migration predominantly through NF-кB/eNOS/NO signaling pathway, which indicates a potential direction for clinical therapy in vascular diseases.]]> W Chiu, T Chiou, A Chiang 2012-04-10T14:24:52Z doi:10.1042/BJ20111383 Portland Press Limited 2012-04-10 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112225 Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV-infected) patients, and (ii) known to preferentially colonize tumor tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a thymidine phosphorylase (TP) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyzes the phosphorolysis of thymidine (K_m = 473 µM) and deoxyuridine (K_m = 578 µM), it prefers uridine (K_m = 92 µM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the nucleoside phosphorylase (NP)-II class pyrimidine nucleoside phosphorylases (PyNP), and is distinct from the NP-II class TP and NP-I class uridine phosphorylases (UP). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.]]> J Vande Voorde, F Gago, K Vrancken, S Liekens, J Balzarini 2012-04-04T14:17:03Z doi:10.1042/BJ20112225 Portland Press Limited 2012-04-04 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120183 S-domains and unsulphated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments. Ultracentrifugation showed that FH formed up to 63% of well-defined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constant K_D of about 0.5 µM. FH structures that are bivalently cross-linked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH-heparin oligomers. The X-ray radius of gyration R_G of FH in the presence of heparin fragments 18 to 36 monosaccharide units long increased significantly from 10.4 to 11.7 nm, and the maximum lengths of FH increased from 35 nm to 40 nm, confirming that large compact oligomers had formed. Surface plasmon resonance of immobilised heparin with full-length FH gave K_D values of 1-3 µM, and similar but weaker K_D values of 4-20 µM for the SCR-6/8 and SCR-16/20 fragments, confirming co-operativity between the two binding sites. The use of minimally-sulphated heparan sulphate fragments that correspond largely to NA-domains showed much weaker binding, proving the importance of S-domains for this interaction. This bivalent and co-operative model of FH binding to heparan sulphate provided novel insights on the immune function of FH at host cell surfaces.]]> S Khan, R Nan, J Gor, B Mulloy, S J Perkins 2012-04-03T13:30:02Z doi:10.1042/BJ20120183 Portland Press Limited 2012-04-03 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112019 C O Brown, K Salem, B A Wagner, S Bera, N Singh, A Tiwari, A Choudhury, G R Buettner, A Goel 2012-04-03T15:05:00Z doi:10.1042/BJ20112019 Portland Press Limited 2012-04-03 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111932 in vitro. However, stimulation with the cAMP-elevating agent forskolin and the beta-adrenergic receptor agonist CL 316,243 resulted in a PKA-dependent phosphorylation and 14-3-3 binding of SIK2. Phosphopeptide mapping of SIK2 revealed several sites phosphorylated in response to cAMP-induction, including Ser358. Site-directed mutagenesis demonstrated that phosphorylation of Ser358, but not the previously reported PKA site Ser587, was required for 14-3-3 binding. Immunocytochemistry illustrated that the localisation of exogenously expressed SIK2 in HEK293 cells was exclusively cytosolic and remained unchanged after cAMP elevation. Fractionation of adipocytes however, revealed a significant increase of wild type, but not Ser358Ala, HA-SIK2 in the cytosol and a concomitant decrease in a particulate fraction after CL 316,243 treatment. This supports a phosphorylation-dependent re-localisation in adipocytes. We hypothesize that regulation of SIK2 by cAMP could play a role for the critical effects of this second messenger on lipid metabolism in adipocytes.]]> E Henriksson, H A Jones, K Patel, M Peggie, N A Morrice, K Sakamoto, O Goransson 2012-03-30T13:39:05Z doi:10.1042/BJ20111932 Portland Press Limited 2012-03-30 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112200 Escherichia coli with exogenously added heme at elevated temperature in the presence of a relatively high concentration of sodium chloride. Kinetic analysis of iron oxidation by E. coli BFR preparations containing varying amounts of heme revealed that heme bound to BFR decreases the rate of iron oxidation at the dinuclear iron ferroxidase sites but increases the rate of iron core formation. Similar kinetic analysis of BFR reconstituted with cobalt-heme revealed that this heme derivative has no influence on the rate of iron core formation. These observations argue that heme bound to E. coli BFR accelerates iron core formation by an electron transfer-based mechanism.]]> S G. Wong, R Abdulqadir, N E Le Brun, G R Moore, A Grant Mauk 2012-03-30T10:09:09Z doi:10.1042/BJ20112200 Portland Press Limited 2012-03-30 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120294 A R Diers, K A Broniowska, C Chang, N Hogg 2012-03-30T09:49:50Z doi:10.1042/BJ20120294 Portland Press Limited 2012-03-30 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120197 Shewanella oneidensis MR-1 identifies three low-spin His/His coordinated c-hemes and a single high-spin c-heme with His/H₂O coordination lying adjacent to the quinol binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (ca. -240 mV) and low-spin (ca. -110, -190 and -265 mV) hemes that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (E_m -80 mV) in the presence of NADH (E_m -320 mV) and an NADH:menadione oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H₂ oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-heme cofactor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc₃. The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.]]> S J Marritt, T G Lowe, J Bye, D G G McMillan, L Shi, J Fredrickson, J Zachara, D J Richardson, M R Cheesman, L J C Jeuken, J N Butt 2012-03-29T14:13:17Z doi:10.1042/BJ20120197 Portland Press Limited 2012-03-29 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111742 D H. Welner, S Lindemose, J Grossmann, N Erik Møllegaard, A N. Olsen, C Helgstrand, K Skriver, L Lo Leggio 2012-03-29T10:13:12Z doi:10.1042/BJ20111742 Portland Press Limited 2012-03-29 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120205 cis-site responsible for its up regulation by DNAJB6 revealed the presence of two binding sites for a transcriptional repressor, MSX1 (muscle segment homeobox gene). Our investigations showed that MSX1 binds the DKK1 promoter and inhibits DKK1 transcription. Interestingly, silencing DNAJB6 resulted in up-regulation of MSX1 concomitant with increased stabilization of β-catenin. ChIP studies revealed that β-catenin binds MSX1 promoter and stabilization of β-catenin elevates MSX1 transcription, indicating that β-catenin works as a transcription co-activator for MSX1. Functionally, exogenous expression of MSX1 in DNAJB6 expressing cells promotes the mesenchymal phenotype by suppression of DKK1. Thus we have identified a novel regulatory mechanism of DNAJB6 mediated DKK1 transcription up-regulation that can influence epithelial-mesenchymal transition. DKK1 is a feedback regulator of β-catenin levels. Thus our studies also define an additional negative control of this β-catenin-DKK1 feedback loop by MSX1, which may potentially contribute to excessive stabilization of β-catenin.]]> M E. Menezes, A Mitra, L A. Shevde, R S Samant 2012-03-28T14:07:02Z doi:10.1042/BJ20120205 Portland Press Limited 2012-03-28 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111890 X Dong, X Fu, S Fan, P Guo, D Su, J Dong 2012-03-28T10:46:12Z doi:10.1042/BJ20111890 Portland Press Limited 2012-03-28 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111268 O Pekar, S Benjamin, H Weidberg, S Smaldone, F Ramirez, M Horowitz 2012-03-26T14:31:46Z doi:10.1042/BJ20111268 Portland Press Limited 2012-03-26 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120163 (i) Augmented flux through the TCA cycle, as evidenced by larger incorporation of ¹³C into the cycle (anaplerosis) and increased generation of ¹³C isotopomers of citrate, glutamate and aspartate (cataplerosis); (ii) Lowering of hepatic [Pi] and elevated [ATP], denoting greater phosphorylation potential and energy state; (iii) Stimulation of glycogen synthesis from glucose but inhibition of glycogen synthesis from 3-carbon precursors; (iv) Increased synthesis of N-acetlylglutamate and consequently augmented ureagenesis; (v) Increased synthesis of glutamine, alanine, serine and glycine; and (vi) Increased production and outflow of lactate. The current study provides a deeper insight into the hepatic actions of GKAs and uncovers the potential benefits and risks of GKA for treatment of diabetes. GKA improved hepatic bioenergetics, ureagenesis and glycogenesis but decreased gluconeogenesis with a potential risk of lactic acidosis and fatty liver.]]> I Nissim, O Horyn, I Nissim, Y Daikhin, S L. Wehrli, M Yudkoff, F M. Matschinsky 2012-03-26T13:56:40Z doi:10.1042/BJ20120163 Portland Press Limited 2012-03-26 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112032 Pseudomonas aeruginosa contains over 20 genes encoding predicted hotdog-fold proteins, none of which have been experimentally characterized. We have found that two P. aeruginosa hotdog proteins display high thioesterase activity against 3-hydroxy-3-methylglutaryl-CoA and glutaryl-CoA (PA5202), and octanoyl-CoA (PA2801). Crystal structures of these proteins were solved (1.70 and 1.75 Å) and revealed a hotdog fold with a potential catalytic carboxylate residue located on the long alpha helix (Asp57 in PA5202 and Glu35 in PA2801). Alanine replacement mutagenesis of PA5202 identified four residues (Asn42, Arg43, Asp57, and Thr76), which are critical for activity and are located in the active site. A P. aeruginosa PA5202 deletion strain showed an increased secretion of the antimicrobial pigment pyocyanine and an increased expression of genes involved in pyocyanin biosynthesis suggesting a functional link between the PA5202 activity and pyocyanin production. Thus, the P. aeruginosa hotdog thioesterases PA5202 and PA2801 have similar structures, but exhibit different substrate preferences and functions.]]> C Gonzalez, A Tchigvintsev, G Brown, R Flick, E Evdokimova, X Xu, J Osipiuk, M Cuff, S Lynch, A Joachimiak, A Savchenko, A F. Yakunin 2012-03-23T10:59:46Z doi:10.1042/BJ20112032 Portland Press Limited 2012-03-23 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112141 A M Thackray, F Muhammad, C Zhang, Y Di, T R Jahn, M Landgraf, D C Crowther, J Felix Evers, R Bujdoso 2012-03-21T15:14:55Z doi:10.1042/BJ20112141 Portland Press Limited 2012-03-21 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120036 Bryum coronatum, is an endo-acting enzyme that hydrolyzes glycosidic bonds of chitin, a β-1,4-linked polysacharide of N-acetylglucosamine, (GlcNAc)_n, through an inverting mechanism. When the wild-type enzyme was incubated with alpha-(GlcNAc)₂ fluoride [alpha-(GlcNAc)₂-F] in the absence or presence of (GlcNAc)₂, (GlcNAc)₂ and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. (J. Biol. Chem. 281, 1426-1431 (2006); Glycobiology, 18, 325-330 (2008)); that is, Ser102 holding a nucleophilic water molecule and Glu70 acting as a catalytic base were mutated, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G, and E70Q. In all mutated enzymes except S102T, hydrolytic activity toward (GlcNAc)₆ was not detected under the conditions we employed. Among the inactive BcChi-A mutants, S102A, S102C, S102G, and E70G were found to successfully synthesize (GlcNAc)₄ as a major product from alpha-(GlcNAc)₂-F in the presence of (GlcNAc)₂. The S102A mutant showed the greatest glycosynthase activity due to its enhanced F^- releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.]]> T Ohnuma, T Fukuda, S Dozen, Y Honda, M Kitaoka, T Fukamizo 2012-03-21T14:14:03Z doi:10.1042/BJ20120036 Portland Press Limited 2012-03-21 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111811 Solanum tuberosum with myo-[2-³H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone/receptor complexes. Our work affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.]]> D E Hanke, P N Parmar, S EK Caddick, P Green, C A Brearley 2012-03-20T11:44:43Z doi:10.1042/BJ20111811 Portland Press Limited 2012-03-20 BJ Plant http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112219 I C Martins, F Gomes-Neto, A F Faustino, F A Carvalho, F A Carneiro, P T Bozza, R Mohana-Borges, M A Castanho, F C Almeida, N C. Santos, A T Da Poian 2012-03-19T14:50:05Z doi:10.1042/BJ20112219 Portland Press Limited 2012-03-19 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120098 In vitro, the tumor suppressor PTEN displays intrinsic phosphatase activity towards both protein and lipid substrates. In vivo, the lipid phosphatase activity of PTEN, through which it dephosphorylates the 3 position in the inositol sugar of phosphatidylinositol derivatives, is important for its tumor suppressor function; however, the significance of its protein phosphatase activity remains unclear. Utilizing two-photon laser scanning microscopy and biolistic gene delivery of GFP-tagged constructs into organotypic hippocampal slice cultures, we have developed an assay of PTEN function in living tissue. Using this bioassay, we have demonstrated that overexpression of wild-type PTEN led to a decrease in spine density in neurons. Furthermore, it was the protein phosphatase activity, but not the lipid phosphatase activity, of PTEN that was essential for this effect. The ability of PTEN to decrease neuronal spine density depended upon the phosphorylation status of Ser and Thr residues in its C-terminal segment and the integrity of the C-terminal PDZ-binding motif. Our studies reveal a new aspect of the function of this important tumor suppressor and suggest that in addition to dephosphorylating the 3 position in phosphatidylinositol phospholipids, the critical protein substrate of PTEN may be PTEN itself.]]> X Catherine Zhang, A Piccini, M P. Myers, L Van Aelst, N K Tonks 2012-03-13T12:31:48Z doi:10.1042/BJ20120098 Portland Press Limited 2012-03-13 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112079 S Geula, D Ben-Hail, V Shoshan-Barmatz 2012-03-08T12:29:12Z doi:10.1042/BJ20112079 Portland Press Limited 2012-03-08 BJ Structure