http://www.biochemj.org Biochemical Journal RSS Feed 0264-6021 1470-8728 Biochemical Journal http://www.biochemj.org/images/bj_Name.gif http://www.biochemj.org http://www.biochemj.org/bj/442/0001/bj4420001.htm IDPs (intrinsically disordered proteins) are highly abundant in eukaryotic proteomes and important for cellular functions, especially in cell signalling and transcriptional regulation. An IDR (intrinsically disordered region) within an IDP often undergoes disorder-to-order transitions upon binding to various partners, allowing an IDP to recognize and bind different partners at various binding interfaces. Plant-specific GRAS proteins play critical and diverse roles in plant development and signalling, and act as integrators of signals from multiple plant growth regulatory and environmental inputs. Possessing an intrinsically disordered N-terminal domain, the GRAS proteins constitute the first functionally required unfoldome from the plant kingdom. Furthermore, the N-terminal domains of GRAS proteins contain MoRFs (molecular recognition features), short interaction-prone segments that are located within IDRs and are able to recognize their interacting partners by undergoing disorder-to-order transitions upon binding to these specific partners. These MoRFs represent potential protein-protein binding sites and may be acting as molecular bait in recognition events during plant development. Intrinsic disorder provides GRAS proteins with a degree of binding plasticity that may be linked to their functional versatility. As an overview of structure-function relationships for GRAS proteins, the present review covers the main biological functions of the GRAS family, the IDRs within these proteins and their implications for understanding mode-of-action.

]]> Xiaolin Sun, William T. Jones and Erik H. A. Rikkerink 2012-02-15 doi:10.1042/BJ20111766 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0013/bj4420013.htm Post-translational modifications are used by cells to link additional information to proteins. Most modifications are subtle and concern small moieties such as a phosphate group or a lipid. In contrast, protein ubiquitylation entails the covalent attachment of a full-length protein such as ubiquitin. The protein ubiquitylation machinery is remarkably complex, comprising more than 15 Ubls (ubiquitin-like proteins) and several hundreds of ubiquitin-conjugating enzymes. Ubiquitin is best known for its role as a tag that induces protein destruction either by the proteasome or through targeting to lysosomes. However, addition of one or more Ubls also affects vesicular traffic, protein-protein interactions and signal transduction. It is by now well established that ubiquitylation is a component of most, if not all, cellular signalling pathways. Owing to its abundance in controlling cellular functions, ubiquitylation is also of key relevance to human pathologies, including cancer and inflammation. In the present review, we focus on its role in the control of cell adhesion, polarity and directional migration. It will become clear that protein modification by Ubls occurs at every level from the receptors at the plasma membrane down to cytoskeletal components such as actin, with differential consequences for the pathway's final output. Since ubiquitylation is fast as well as reversible, it represents a bona fide signalling event, which is used to fine-tune a cell's responses to receptor agonists.

]]> Antje Schaefer, Micha Nethe and Peter L. Hordijk 2012-02-15 doi:10.1042/BJ20111815 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0027/bj4420027.htm Newly determined crystal structures of the photosynthetic RC (reaction centre) from two substrains of the non-sulfur purple bacterium Blastochloris viridis strain DSM 133, together with analysis of their gene sequences, has revealed intraspecies evolutionary changes over a period of 14 years. Over 100 point mutations were identified between these two substrains in the four genes encoding the protein subunits of the RC, of which approximately one-fifth resulted in a total of 16 amino acid changes. The most interesting difference was in the M subunit where the change from a leucine residue to glycine in the carotenoid-binding pocket allowed NS5 (1,2-dihydroneurosporene) to adopt a more sterically favoured conformation, similar to the carotenoid conformation found in other related RCs. The results of the present study, together with a high rate of mutations in laboratory bacterial cultures described recently, suggest that bacteria evolve faster than has been generally recognized. The possibility that amino acid changes occur within protein sequences, without exhibiting any immediately observable phenotype, should be taken into account in studies that involve long-term continuous growth of pure bacterial cultures. The Blc. viridis RC is often studied with sophisticated biophysical techniques and changes such as those described here may well affect their outcome. In other words, there is a danger that laboratory-to-laboratory variation could well be due to different groups not realising that they are actually working with slightly different proteins. A way around this problem is suggested.

]]> Aleksander W. Roszak, Vladimíra Moulisová, Adhie D. P. Reksodipuro, Alastair T. Gardiner, Ritsuko Fujii, Hideki Hashimoto, Neil W. Isaacs and Richard J. Cogdell 2012-02-15 doi:10.1042/BJ20111540 Blastochloris viridis: evolution in the laboratory]]> Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0039/bj4420039.htm HD (Huntington's disease) is caused by an expanded polyQ (polyglutamine) repeat in the htt (huntingtin protein). GABAergic medium spiny neurons in the striatum are mostly affected in HD. However, mhtt (mutant huntingtin)-induced molecular changes in these neurons remain largely unknown. The present study focuses on the effect of mhtt on the subcellular localization of GAD (glutamic acid decarboxylase), the enzyme responsible for synthesizing GABA (γ-aminobutyric acid). We report that the subcellular distribution of GAD is significantly altered in two neuronal cell lines that express either the N-terminus of mhtt or full-length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal htt; however, it diffuses in the cytosol of cells expressing mhtt. As a result, vesicle-associated GAD65 trafficking is impaired. Since palmitoylation of GAD65 is required for GAD65 trafficking, we then demonstrate that palmitoylation of GAD65 is reduced in the HD model. Furthermore, overexpression of HIP14 (huntingtin-interacting protein 14), the enzyme responsible for palmitoylating GAD65 in vivo, could rescue GAD65 palmitoylation and vesicle-associated GAD65 trafficking. Taken together, our data support the idea that GAD65 palmitoylation is important for the delivery of GAD65 to inhibitory synapses and suggest that impairment of GAD65 palmitoylation by mhtt may lead to altered inhibitory neurotransmission in HD.

]]> Daniel B. Rush, Rebecca T. Leon, Mark H. McCollum, Ryan W. Treu and Jianning Wei 2012-02-15 doi:10.1042/BJ20110679 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0049/bj4420049.htm Our recent studies suggest that H₂ (hydrogen) has a potential as a novel radioprotector without known toxic side effects. The present study was designed to examine the underlying radioprotective mechanism of H₂ and its protective role on irradiated germ cells. Produced by the Fenton reaction and radiolysis of H₂O, hydroxyl radicals (^•OH) were identified as the free radical species that were reduced by H₂. We used a H₂ microelectrode to dynamically detect H₂ concentration in vivo, and found H₂ significantly reduced in situ fluorescence intensity of hydroxyphenyl fluorescein; however, as we treated the mice with H₂ after irradiation, the decrease is not significant. We found that pre-treatment of H₂ to IR (ionizing radiation) significantly suppressed the reaction of ^•OH and the cellular macromolecules which caused lipid peroxidation, protein carbonyl and oxidatively damaged DNA. The radioprotective effect of H₂ on male germ cells was supported by ameliorated apoptotic findings examined by morphological changes and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) in testicular tissue, and by preserved viability of stem spermatogonia examined for testicular histological parameters, daily sperm production and sperm quality; we used WR-2721 [S-2-(3-aminopropylamino)ethyl phosphorothioic acid] as a reference compound. Our results represent the first in vivo evidence in support of a radioprotective role of H₂ by neutralizing ^•OH in irradiated tissue with no side effects.

]]> Yunhai Chuai, Fu Gao, Bailong Li, Luqian Zhao, Liren Qian, Fei Cao, Lei Wang, Xuejun Sun, Jianguo Cui and Jianming Cai 2012-02-15 doi:10.1042/BJ20111786 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0057/bj4420057.htm The uptake of potassium ions (K⁺) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2-2.5 pH units) is perceived by voltage-gated inward potassium channels (K_in) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal K_in channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal K_in channel KAT1, mutations in the unique histidine exposed to the solvent (His²⁶⁷) do not affect the pH dependency. We demonstrate in the present study that His²⁶⁷ of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe²⁶⁶ shifts its pK_a to undetectable values through a cation-π interaction. Instead, we show that Glu²⁴⁰ placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu²⁴⁰, several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel.

]]> Wendy González, Janin Riedelsberger, Samuel E. Morales‑Navarro, Julio Caballero, Jans H. Alzate‑Morales, Fernando D. González‑Nilo and Ingo Dreyer 2012-02-15 doi:10.1042/BJ20111498 +-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids]]> Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0065/bj4420065.htm Transcriptional regulation requires co-ordinated action of transcription factors, co-activator complexes and general transcription factors to access specific loci in the dense chromatin structure. In the present study we demonstrate that the transcriptional co-regulator SPBP [stromelysin-1 PDGF (platelet-derived growth factor)-responsive element binding protein] contains two independent chromatin-binding domains, the SPBP-(1551-1666) region and the C-terminal extended PHD [ePHD/ADD (extended plant homeodomain/ATRX-DNMT3-DNMT3L)] domain. The region 1551-1666 is a novel core nucleosome-interaction domain located adjacent to the AT-hook motif in the DNA-binding domain. This novel nucleosome-binding region is critically important for proper localization of SPBP in the cell nucleus. The ePHD/ADD domain associates with nucleosomes in a histone tail-dependent manner, and has significant impact on the dynamic interaction between SPBP and chromatin. Furthermore, SPBP and its homologue RAI1 (retinoic-acid-inducible protein 1), are strongly enriched on chromatin in interphase HeLa cells, and both proteins display low nuclear mobility. RAI1 contains a region with homology to the novel nucleosome-binding region SPBP-(1551-1666) and an ePHD/ADD domain with ability to bind nucleosomes. These results indicate that the transcriptional co-regulator SPBP and its homologue RAI1 implicated in Smith-Magenis syndrome and Potocki-Lupski syndrome both belong to the expanding family of chromatin-binding proteins containing several domains involved in specific chromatin interactions.

]]> Sagar Darvekar, Sylvia Sagen Johnsen, Agnete Bratsberg Eriksen, Terje Johansen and Eva Sjøttem 2012-02-15 doi:10.1042/BJ20111230 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0077/bj4420077.htm DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron-sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.

]]> Anne‑Marie McRobbie, Bjoern Meyer, Christophe Rouillon, Biljana Petrovic‑Stojanovska, Huanting Liu and Malcolm F. White 2012-02-15 doi:10.1042/BJ20111903 Staphylococcus aureus DinG, a helicase that has evolved into a nuclease]]> Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0085/bj4420085.htm The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B.

]]> Corinne Appia‑Ayme, Andrea Hall, Elaine Patrick, Shiny Rajadurai, Thomas A. Clarke and Gary Rowley 2012-02-15 doi:10.1042/BJ20111639 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0095/bj4420095.htm FHC (familial hypertrophic cardiomyopathy) is a heritable form of cardiac hypertrophy caused by mutations in genes encoding sarcomeric proteins. The present study focuses on the A13T mutation in the human ventricular myosin RLC (regulatory light chain) that is associated with a rare FHC variant defined by mid-ventricular obstruction and septal hypertrophy. We generated heart-specific Tg (transgenic) mice with ~10% of human A13T-RLC mutant replacing the endogenous mouse cardiac RLC. Histopathological examinations of longitudinal heart sections from Tg-A13T mice showed enlarged interventricular septa and profound fibrotic lesions compared with Tg-WT (wild-type), expressing the human ventricular RLC, or non-Tg mice. Functional studies revealed an abnormal A13T mutation-induced increase in isometric force production, no change in the force-pCa relationship and a decreased V_max of the acto-myosin ATPase. In addition, a fluorescence-based assay showed a 3-fold lower binding affinity of the recombinant A13T mutant for the RLC-depleted porcine myosin compared with WT-RLC. These results suggest that the A13T mutation triggers a hypertrophic response through changes in cardiac sarcomere organization and myosin cross-bridge function leading to abnormal remodelling of the heart. The significant functional changes observed, despite a low level of A13T mutant incorporation into myofilaments, suggest a 'poison-peptide' mechanism of disease.

]]> Katarzyna Kazmierczak, Priya Muthu, Wenrui Huang, Michelle Jones, Yingcai Wang and Danuta Szczesna‑Cordary 2012-02-15 doi:10.1042/BJ20111145 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0105/bj4420105.htm eEF2K (eukaryotic elongation factor 2 kinase) is a Ca²⁺/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called 'α-kinases' which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured 'linker' region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca²⁺/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

]]> Craig R. Pigott, Halina Mikolajek, Claire E. Moore, Stephen J. Finn, Curtis W. Phippen, Jörn M. Werner and Christopher G. Proud 2012-02-15 doi:10.1042/BJ20111536 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0119/bj4420119.htm The T3SS (type III secretion system) is a multi-protein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. This apparatus spans both bacterial membranes and transports virulence factors from the bacterial cytoplasm into eukaryotic host cells. The T3SS exports substrates in a hierarchical and temporal manner. The first secreted substrates are the rod/needle proteins which are incorporated into the T3SS apparatus and are required for the secretion of later substrates, the translocators and effectors. In the present study, we provide evidence that rOrf8/EscI, a poorly characterized locus of enterocyte effacement-encoded protein, functions as the inner rod protein of the T3SS of EPEC (enteropathogenic Escherichia coli). We demonstrate that EscI is essential for type III secretion and is also secreted as an early substrate of the T3SS. We found that EscI interacts with EscU, the integral membrane protein that is linked to substrate specificity switching, implicating EscI in the substrate-switching event. Furthermore, we showed that EscI self-associates and interacts with the outer membrane secretin EscC, further supporting its function as an inner rod protein. Overall, the results of the present study suggest that EscI is the YscI/PrgJ/MxiI homologue in the T3SS of attaching and effacing pathogens.

]]> Neta Sal‑Man, Wanyin Deng and B. Brett Finlay 2012-02-15 doi:10.1042/BJ20111620 Escherichia coli]]> Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0127/bj4420127.htm Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus Aspergillus fumigatus in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC PLA₂ (phospholipase A₂) [also known as cPLA₂γ (cytosolic PLA₂γ) or PLA2G4C]. Our results infer that A. fumigatus extract can induce cPLA₂γ levels directly in eosinophils, whereas induction in lung epithelial cells is most likely to be a consequence of TNFα (tumour necrosis factor α) secretion by A. fumigatus-activated macrophages. The mechanism of TNFα-dependent induction of cPLA₂γ gene expression was elucidated through a combination of promoter deletions, ChIP (chromatin immunoprecipitation) and overexpression studies in human bronchoepithelial cells, leading to the identification of functionally relevant CRE (cAMP-response element), NF-κB (nuclear factor κB) and E-box promoter elements. ChIP analysis demonstrated that RNA polymerase II, ATF-2 (activating transcription factor 2)-c-Jun, p65-p65 and USF (upstream stimulating factor) 1-USF2 complexes are recruited to the cPLA₂γ enhancer/promoter in response to TNFα, with overexpression and dominant-negative studies implying a strong level of co-operation and interplay between these factors. Overall, our results link cytokine-mediated alterations in cPLA₂γ gene expression with allergic asthma and outline a complex regulatory mechanism.

]]> Justin S. Bickford, Kimberly J. Newsom, John‑David Herlihy, Christian Mueller, Benjamin Keeler, Xiaolei Qiu, Jewell N. Walters, Nan Su, Shannon M. Wallet, Terence R. Flotte and Harry S. Nick 2012-02-15 doi:10.1042/BJ20111269 2 in allergic asthma: transcriptional regulation by TNFα in bronchoepithelial cells]]> Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0139/bj4420139.htm Phosphorylation of the BH3 (Bcl-2 homology domain 3)-only protein BAD (Bcl-2/Bcl-X_L-antagonist, causing cell death) can either directly disrupt its association with the pro-survival proteins Bcl-X_L and/or Bcl-2, or cause association of BAD with 14-3-3 proteins. In the present study, we further characterize phosphorylation of BAD at Ser¹⁷⁰, a unique site with unclear function. We provide further evidence that mutation of Ser¹⁷⁰ to a phospho-mimetic aspartic acid residue (S170D) can have a profound inhibitory effect on the pro-apoptosis function of BAD. Furthermore, mutated BAD with an alanine substitution inhibited cell proliferation, slowing progression specifically through S-phase. We identify the kinase responsible for phosphorylation at this site as CaMKII-γ (γ isoform of Ca²⁺/calmodulin-dependent kinase II), but not the other three isoforms of CaMKII, revealing an extraordinary specificity among these closely related kinases. Furthermore, cytokine treatment increased BAD-Ser¹⁷⁰-directed CaMKII-γ activity and phosphorylation of CaMKII-γ at an activating site, and CaMKII activity directed to the BAD-Ser¹⁷⁰ site was elevated during S-phase. Treating cells with a selective inhibitor of CaMKII caused apoptosis in cells expressing BAD, but not in cells expressing the BAD-S170D mutant. The present study provides support for BAD-Ser¹⁷⁰ phosphorylation playing a key role not only in regulating BAD's pro-apoptotic activity, but also in cell proliferation.

]]> Payman Hojabrpour, Ivan Waissbluth, Mazyar Ghaffari, Michael E. Cox and Vincent Duronio 2012-02-15 doi:10.1042/BJ20111256 170 to regulate cytokine-dependent survival and proliferation]]> Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0151/bj4420151.htm The PI3K (phosphoinositide 3-kinase) pathway is commonly activated in cancer as a consequence of inactivation of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), a major negative regulator of PI3K signalling. In line with this important role of PTEN, mice that are heterozygous for a PTEN-null allele (PTEN^+/− mice) spontaneously develop a variety of tumours in multiple organs. PTEN is a phosphatase with selectivity for PtdIns(3,4,5)P₃, which is produced by the class I isoforms of PI3K (p110α, p110β, p110γ and p110δ). Previous studies indicated that PTEN-deficient cancer cell lines mainly depend on p110β, and that p110β, but not p110α, controls mouse prostate cancer development driven by PTEN loss. In the present study, we investigated whether the ubiquitously expressed p110α can also functionally interact with PTEN in cancer. Using genetic mouse models that mimic systemic administration of p110α- or p110β-selective inhibitors, we confirm that inactivation of p110β, but not p110α, inhibits prostate cancer development in PTEN^+/− mice, but also find that p110α inactivation protects from glomerulonephritis, pheochromocytoma and thyroid cancer induced by PTEN loss. This indicates that p110α can modulate the impact of PTEN loss in disease and tumourigenesis. In primary and immortalized mouse fibroblast cell lines, both p110α and p110β controlled steady-state PtdIns(3,4,5)P₃ levels and Akt signalling induced by heterozygous PTEN loss. In contrast, no correlation was found in primary mouse tissues between PtdIns(3,4,5)P₃ levels, PI3K/PTEN genotype and cancer development. Taken together, our results from the present study show that inactivation of either p110α or p110β can counteract the impact of PTEN inactivation. The potential implications of these findings for PI3K-targeted therapy of cancer are discussed.

]]> Inma M. Berenjeno, Julie Guillermet‑Guibert, Wayne Pearce, Alexander Gray, Stewart Fleming and Bart Vanhaesebroeck 2012-02-15 doi:10.1042/BJ20111741 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0161/bj4420161.htm In in vitro studies class-I PI3Ks (phosphoinositide 3-kinases), class-II PI3Ks and mTOR (mammalian target of rapamycin) have all been described as having roles in the regulation of glucose metabolism. The relative role each plays in the normal signalling processes regulating glucose metabolism in vivo is less clear. Knockout and knockin mouse models have provided some evidence that the class-I PI3K isoforms p110α, p110β, and to a lesser extent p110γ, are necessary for processes regulating glucose metabolism and appetite. However, in these models the PI3K activity is chronically reduced. Therefore we analysed the effects of acutely inhibiting PI3K isoforms alone, or PI3K and mTOR, on glucose metabolism and food intake. In the present study impairments in glucose tolerance, insulin tolerance and increased hepatic glucose output were observed in mice treated with the pan-PI3K/mTOR inhibitors PI-103 and NVP-BEZ235. The finding that ZSTK474 has similar effects indicates that these effects are due to inhibition of PI3K rather than mTOR. The p110α-selective inhibitors PIK75 and A66 also induced these phenotypes, but inhibitors of p110β, p110δ or p110γ induced only minor effects. These drugs caused no significant effects on BMR (basal metabolic rate), O₂ consumption or water intake, but BEZ235, PI-103 and PIK75 did cause a small reduction in food consumption. Surprisingly, pan-PI3K inhibitors or p110α inhibitors caused reductions in animal movement, although the cause of this is not clear. Taken together these studies provide pharmacological evidence to support a pre-eminent role for the p110α isoform of PI3K in pathways acutely regulating glucose metabolism.

]]> Greg C. Smith, Wee Kiat Ong, Gordon W. Rewcastle, Jackie D. Kendall, Weiping Han and Peter R. Shepherd 2012-02-15 doi:10.1042/BJ20111913 in vivo]]> Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0171/bj4420171.htm LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is an endothelial scavenger receptor that is important for the uptake of OxLDL (oxidized low-density lipoprotein) and contributes to the pathogenesis of atherosclerosis. However, the precise structural motifs of OxLDL that are recognized by LOX-1 are unknown. In the present study, we have identified products of lipid peroxidation of OxLDL that serve as ligands for LOX-1. We used CHO (Chinese-hamster ovary) cells that stably express LOX-1 to evaluate the ability of BSA modified by lipid peroxidation to compete with AcLDL (acetylated low-density lipoprotein). We found that HNE (4-hydroxy-2-nonenal)-modified proteins most potently inhibited the uptake of AcLDL. On the basis of the findings that HNE-modified BSA and oxidation of LDL resulted in the formation of HNE-histidine Michael adducts, we examined whether the HNE-histidine adducts could serve as ligands for LOX-1. The authentic HNE-histidine adduct inhibited the uptake of AcLDL in a dose-dependent manner. Furthermore, we found the interaction of LOX-1 with the HNE-histidine adduct to have a dissociation constant of 1.22×10⁻⁸ M using a surface plasmon resonance assay. Finally, we showed that the HNE-histidine adduct stimulated the formation of reactive oxygen species and activated extracellular-signal-regulated kinase 1/2 and NF-κB (nuclear factor κB) in HAECs (human aortic endothelial cells); these signals initiate endothelial dysfunction and lead to atherosclerosis. The present study provides intriguing insights into the molecular details of LOX-1 recognition of OxLDL.

]]> Miyuki Kumano‑Kuramochi, Yuuki Shimozu, Chika Wakita, Mayumi Ohnishi‑Kameyama, Takahiro Shibata, Shigeru Matsunaga, Yuko Takano‑Ishikawa, Jun Watanabe, Masao Goto, Qiuhong Xie, Shiro Komba, Koji Uchida and Sachiko Machida 2012-02-15 doi:10.1042/BJ20111029 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0181/bj4420181.htm The cytosolic GST (glutathione transferase) superfamily has been annotated in the Drosophila melanogaster genome database. Of 36 genes, four undergo alternative splicing to yield a total of 41 GST proteins. In the present study, we have obtained the 41 transcripts encoding proteins by RT (reverse transcription)-PCR using RNA template from Drosophila S2 cells, an embryonic cell line. This observation suggests that all of the annotated DmGSTs (D. melanogaster GSTs) in the proteome are expressed in the late embryonic stages of D. melanogaster. To avoid confusion in naming these numerous DmGSTs, we have designated them following the universal GST nomenclature as well as previous designations that fit within this classification. Furthermore, in the cell line, we identified an apparent processed pseudogene, gste8, in addition to two isoforms from the Delta class that have been published previously. Only approximately one-third of the expressed DmGSTs could be purified by conventional GSH affinity chromatography. The diverse kinetic properties as well as physiological substrate specificity of the DmGSTs are such that each individual enzyme displayed a unique character even compared with members from the same class.

]]> Chonticha Saisawang, Jantana Wongsantichon and Albert J. Ketterman 2012-02-15 doi:10.1042/BJ20111747 Drosophila melanogaster]]> Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0191/bj4420191.htm S-nitrosothiols are products of nitric oxide (NO) metabolism that have been implicated in a plethora of signalling processes. However, mechanisms of S-nitrosothiol formation in biological systems are uncertain, and no efficient protein-mediated process has been identified. Recently, we observed that ferric cytochrome c can promote S-nitrosoglutathione formation from NO and glutathione by acting as an electron acceptor under anaerobic conditions. In the present study, we show that this mechanism is also robust under oxygenated conditions, that cytochrome c can promote protein S-nitrosation via a transnitrosation reaction and that cell lysate depleted of cytochrome c exhibits a lower capacity to synthesize S-nitrosothiols. Importantly, we also demonstrate that this mechanism is functional in living cells. Lower S-nitrosothiol synthesis activity, from donor and nitric oxide synthase-generated NO, was found in cytochrome c-deficient mouse embryonic cells as compared with wild-type controls. Taken together, these data point to cytochrome c as a biological mediator of protein S-nitrosation in cells. This is the most efficient and concerted mechanism of S-nitrosothiol formation reported so far.

]]> Katarzyna A. Broniowska, Agnes Keszler, Swati Basu, Daniel B. Kim‑Shapiro and Neil Hogg 2012-02-15 doi:10.1042/BJ20111294 c-mediated formation of S-nitrosothiol in cells]]> Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0199/bj4420199.htm Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3′UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3′UTR that may act as regulatory sequences. Analysis of ODC 3′UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development.

]]> Sofia Origanti, Shannon L. Nowotarski, Theresa D. Carr, Suzanne Sass‑Kuhn, Lan Xiao, Jian‑Ying Wang and Lisa M. Shantz 2012-02-15 doi:10.1042/BJ20111464 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0209/bj4420209.htm The retromer complex is a conserved endosomal protein sorting complex that sorts membrane proteins into nascent endosomal tubules. The recognition of membrane proteins is mediated by the cargo-selective retromer complex, a stable trimer of the Vps35 (vacuolar protein sorting 35), Vps29 and Vps26 proteins. We have recently reported that the cargo-selective retromer complex associates with the WASH (Wiskott-Aldrich syndrome homologue) complex, a multimeric protein complex that regulates tubule dynamics at endosomes. In the present study, we show that the retromer-WASH complex interaction occurs through the long unstructured 'tail' domain of the WASH complex-Fam21 protein binding to Vps35, an interaction that is necessary and sufficient to target the WASH complex to endosomes. The Fam21-tail also binds to FKBP15 (FK506-binding protein 15), a protein associated with ulcerative colitis, to mediate the membrane association of FKBP15. Elevated Fam21-tail expression inhibits the association of the WASH complex with retromer, resulting in increased cytoplasmic WASH complex. Additionally, overexpression of the Fam21-tail results in cell-spreading defects, implicating the activity of the WASH complex in regulating the mobilization of membrane into the endosome-to-cell surface pathway.

]]> Michael E. Harbour, Sophia Y. Breusegem and Matthew N. J. Seaman 2012-02-15 doi:10.1042/BJ20111761 Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0221/bj4420221.htm Non-enzymatic glycation is a complex series of reactions between reducing sugars and amino groups of proteins. Accumulation of AGEs (advanced glycation end-products) due to non-enzymatic glycation has been related to several diseases associated with aging and diabetes. The formation of AGEs is accelerated in hyperglycaemic conditions, which alters the structure and function of long-lived proteins, thereby contributing to long-term diabetic complications. The present study describes AGE inhibition and the mechanism of action of a new antiglycating agent, EA (ellagic acid), a flavonoid present in many dietary sources. Inhibition of AGE formation by EA was demonstrated with different proteins, namely eye lens TSP (total soluble protein), Hb (haemoglobin), lysozyme and BSA, using different glycating agents such as fructose, ribose and methylglyoxal by a set of complementary methods. These results suggest that the antiglycating action of EA seems to involve, apart from inhibition of a few fluorescent AGEs, predominantly inhibition of CEL [N^ϵ-(carboxyethyl)lysine] through scavenging of the dicarbonyl compounds. Furthermore, MALDI-TOF-MS (matrix-assisted laser-desorption ionisation-time-of-flight MS) analysis confirms inhibition of the formation of CEL on lysozyme on in vitro glycation by EA. Prevention of glycation-mediated β-sheet formation in Hb and lysozyme by EA confirm its antiglycating ability. Inhibition of glycosylated Hb formation in human blood under ex vivo high-glucose conditions signifies the physiological antiglycating potential of EA. We have also determined the effectiveness of EA against loss of eye lens transparency through inhibition of AGEs in the lens organ culture system. These findings establish the antiglycating potential of EA and its in vivo utility in controlling AGE-mediated diabetic pathologies.

]]> Puppala Muthenna, Chandrasekhar Akileshwari and G. Bhanuprakash Reddy 2012-02-15 doi:10.1042/BJ20110846 N^ϵ-(carboxymethyl)lysine]]> Portland Press Ltd. 2012-02-15 http://www.biochemj.org/bj/442/0231/bj4420231.htm NM (nemaline myopathy) is a rare genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding β-Tm (tropomyosin). Tm controls muscle contraction by inhibiting actin-myosin interaction in a calcium-sensitive manner. In the present study, we have investigated the pathogenetic mechanisms underlying five disease-causing mutations in Tm. We show that four of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, whereas two show defective Ca²⁺ activation of contractility. We have also mapped the amino acids altered by the mutation to regions important for actin binding and note that two of the mutations cause altered protein conformation, which could account for impaired actin affinity.

]]> Minttu Marttila, Elina Lemola, William Wallefeld, Massimiliano Memo, Kati Donner, Nigel G. Laing, Steven Marston, Mikaela Grönholm and Carina Wallgren‑Pettersson 2012-02-15 doi:10.1042/BJ20111030 TPM2-related nemaline and cap myopathy]]> Portland Press Ltd. 2012-02-15