http://www.biochemj.org Biochemical Journal RSS feed -- BJ Gene 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=BJ20130432 G Shankarling, K W Lynch 2013-05-07T11:21:40Z doi:10.1042/BJ20130432 Portland Press Limited 2013-05-07 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20121126 K Han, D E Ayer 2013-05-01T09:39:43Z doi:10.1042/BJ20121126 Portland Press Limited 2013-05-01 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20121598 H Kim, E Bae, D Jeong, M Kim, W Jin, S Park, G Han, G Carman, E Koh, K Kim 2013-04-30T09:24:37Z doi:10.1042/BJ20121598 Portland Press Limited 2013-04-30 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20121699 Arabidopsis thaliana modifies the 3'-terminal nucleotides of small regulatory RNAs. Although it is one of the best characterized members of the 2’-O-methyltransferase family, many aspects of its interactions with the cofactor and substrate RNA remained unresolved. To better understand substrate interactions and contributions of individual steps during HEN1 catalysis, we studied binding and methylation kinetics using series of unmethylated, hemimethylated and doubly methylated miRNA and siRNA substrates. Our studies indicate that HEN1 specifically binds double-stranded unmethylated or hemimethylated miR173/miR173* substrates with sub-nanomolar affinity in a cofactor-dependent manner. Kinetic studies under single turnover and pre-steady state conditions in combination with isotope partitioning analysis showed that the binary HEN1•miRNA/miRNA* complex is catalytically competent, however successive methylation of the two strands in a RNA duplex occurs in a non-processive (distributive) manner. We also find that the observed moderate methylation strand preference is largely exerted at the RNA binding step and is fairly independent of the nature of the 3’-terminal nucleobase but shows some dependency on proximal nucleotide mispairs. Our results thus provide novel insights into the mechanism of RNA recognition and modification by a representative small RNA 2’-O-methyltransferase.]]> A Plotnikova, S Baranauskė, A Osipenko, S Klimašauskas, G Vilkaitis 2013-04-29T10:11:32Z doi:10.1042/BJ20121699 Portland Press Limited 2013-04-29 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20130049 Dio3) was identified as a gene that exhibited specific translational repression and had a functional role in a number of relevant canonical pathways. Western blot analysis indicated that this repression led to reduced Dio3 protein (D3) levels, enhanced over time and with increased dose. Using Northern blotting techniques and quantitative RT-PCR we further confirmed that there was no reduction in Dio3 mRNA, suggesting that translational repression of Dio3 is an important determinant of the reduced D3 protein expression following liver damage. Finally, we show that drug-induced hepatotoxicity appears to cause localised disruptions in thyroid hormone levels in the liver and plasma. We suggest that this leads to reduced translation of Dio3 mRNA, which results in decreased D3 production. It may, therefore, be possible that this is an important mechanism by which the liver can, upon early signs of damage, act rapidly to maintain its own energy equilibrium, thereby avoiding global disruption of the hypothalamic-pituitary-thyroid axis.]]> K M Dudek, L Suter, V M Darras, E L Marczylo, T W Gant 2013-04-16T09:56:56Z doi:10.1042/BJ20130049 Portland Press Limited 2013-04-16 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20121908 4. Surprisingly, ALKBH1 also forms a stable protein-DNA adduct in the absence of a reducing agent. Experiments with different substrates demonstrated that the protein covalently binds to the 5' DNA product; i.e., the fragment containing an α,β-unsaturated aldehyde. The amino terminal domain of ALKBH1 was identified as the main site of linkage with DNA. By contrast, mutagenesis studies suggest that the primary catalytic residue forming the imine linkage is Lys133, with Lys154 and other Lys residues in this region serving in opportunistic roles. These findings confirm the classification of ALKBH1 as an AP lyase, identify the primary and a secondary Lys involved in the lyase reaction, and demonstrate the protein forms a covalent adduct with the 5' DNA product. We propose two plausible chemical mechanisms to account for the covalent attachment.]]> T A. Müller, M M. Andrzejak, R P. Hausinger 2013-04-12T11:11:44Z doi:10.1042/BJ20121908 Portland Press Limited 2013-04-12 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20121742 leucine-rich repeat kinase 2 (LRRK2) are a major cause of Parkinson’s disease (PD). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, The Michael J. Fox Foundation for Parkinson’s Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. Herein, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation, and kinase activity assays, in rat, mouse, and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labeling in tissue derived from LRRK2 knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease.]]> P Davies, K M Hinkle, N N Sukar, B Sepulveda, R Mesias, G Serrano, D R Alessi, T G Beach, D L Benson, C L White III, R M Cowell, S S Das, A B West, H L Melrose 2013-04-05T15:31:38Z doi:10.1042/BJ20121742 Portland Press Limited 2013-04-05 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20130012 L Song, C Bowen, E P Gelmann 2013-04-05T10:01:10Z doi:10.1042/BJ20130012 Portland Press Limited 2013-04-05 BJ Biomolecules http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20121277 per se, to stably interact with DNA or NPM1.]]> M Poletto, C Vascotto, P L. Scognamiglio, L Lirussi, D Marasco, G Tell 2013-04-02T09:38:44Z doi:10.1042/BJ20121277 Portland Press Limited 2013-04-02 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20130154 Escherichia coli glycogen metabolism involves regulation of the glgBXCAP operon expression and allosteric control of GlgC-mediated catalysis of ATP and glucose-1-phosphate (G1P) to ADP-glucose linked to glycogen biosynthesis. E. coli glycogen metabolism is also affected by glgS. Though the precise function of the protein it encodes is unknown, its deficiency causes both reduced glycogen content and enhanced levels of the GlgC negative allosteric regulator AMP. Transcriptomic analyses carried out in this work revealed that, compared with their isogenic BW25113 wild type strain, glgS null (ΔglgS) mutants have increased expression of operons involved in the synthesis of type 1 fimbriae adhesins, flagella, and nucleotides. In concordance, ΔglgS cells were hyperflagellated and hyperfimbriated, and displayed elevated swarming motility; these phenotypes were all reverted by ectopic glgS expression. Also, ΔglgS cells accumulated high colanic acid content, and displayed increased ability to form biofilms on polysterene surfaces. F-driven conjugation based large-scale interaction studies of glgS with all the nonessential genes of E. coli showed that deletion of purine biosynthesis genes complement the glycogen-deficient, high motility and high biofilm content phenotypes of ΔglgS cells. Overall, these data indicate that glycogen deficiency in ΔglgS cells can be ascribed to high flagellar propulsion, and high exopolysaccharide and purine nucleotides biosynthetic activites competing with GlgC for the same ATP and G1P pools. Supporting this proposal, glycogen-less ΔglgC cells displayed an elevated swarming motility, and accumulated high levels of colanic acid and biofilm. Furthermore, glgC over-expression reverted the glycogen-deficient, high swarming motility, high colanic acid and high biofilm content phenotypes of ΔglgS cells. Because GlgS emerges now as a major determinant of E. coli surface composition, and because its effect on glycogen metabolism appears to be only indirect, we propose to rename it as ScoR for Surface Composition Regulator.]]> M Rahimpour, M Montero, G Almagro, A M Viale, A Sevilla, M Cánovas, F José Muñoz, E Baroja-Fernández, A Bahaji, G Eydallin, H Dose, R Takeuchi, H Mori, J Pozueta-Romero 2013-03-28T16:39:00Z doi:10.1042/BJ20130154 Portland Press Limited 2013-03-28 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20121666 P C Encarnacao, V P Ramirez, C Zhang, B J Aneskievich 2013-03-07T11:29:00Z doi:10.1042/BJ20121666 Portland Press Limited 2013-03-07 BJ Gene