bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolomic analyses reveal profound differences in glycolytic and tricarboxylic acid cycle metabolism in glucose-responsive and -unresponsive clonal β-cell lines
Authors: Spégel Peter , Malmgren Siri , Sharoyko Vladimir V. , Newsholme Philip , Koeck Thomas , Mulder Hindrik
Published: February 2011

Insulin secretion from pancreatic β-cells is controlled by complex metabolic and energetic changes provoked by exposure to metabolic fuels. Perturbations in these processes lead to impaired insulin secretion, the ultimate cause of T2D (Type 2 diabetes). To increase our understanding of stimulus–secretion coupling and metabolic processes potentially involved in the pathogenesis of T2D, a comprehensive investigation of the metabolic response in the glucose-responsive INS-1 832/13 and glucose-unresponsive INS-1 832/2 β-cell lines was performed. For this metabolomics analysis, we used GC/MS (gas chromatography/mass spectrometry) combined with multivariate statistics. We found that perturbed secretion in the 832/2 line was characterized by disturbed coupling of glycolytic and TCA (tricarboxylic acid)-cycle metabolism. The importance of this metabolic coupling was reinforced by our observation that insulin secretion partially could be reinstated by stimulation of the cells with mitochondrial fuels which bypass glycolytic metabolism. Furthermore, metabolic and functional profiling of additional β-cell lines (INS-1, INS-1 832/1) confirmed the important role of coupled glycolytic and TCA-cycle metabolism in stimulus–secretion coupling. Dependence of the unresponsive clones on glycolytic metabolism was paralleled by increased stabilization of HIF-1α (hypoxia-inducible factor 1α). The relevance of a similar perturbation for human T2D was suggested by increased expression of HIF-1α target genes in islets from T2D patients.
Proteins containing oxidized amino acids induce apoptosis in human monocytes
Authors: Dunlop Rachael A. , Brunk Ulf T. , Rodgers Kenneth J.
Published: February 2011

Cellular deposits of oxidized and aggregated proteins are hallmarks of a variety of age-related disorders, but whether such proteins contribute to pathology is not well understood. We previously reported that oxidized proteins form lipofuscin/ceroid-like bodies with a lysosomal-type distribution and up-regulate the transcription and translation of proteolytic lysosomal enzymes in cultured J774 mouse macrophages. Given the recently identified role of lysosomes in the induction of apoptosis, we have extended our studies to explore a role for oxidized proteins in apoptosis. Oxidized proteins were biosynthetically generated in situ by substituting oxidized analogues for parent amino acids. Apoptosis was measured with Annexin-V/PI (propidium iodide), TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling), MMP (mitochondrial membrane permeabilization), caspase activation and cytochrome c release, and related to lysosomal membrane permeabilization. Synthesized proteins containing the tyrosine oxidation product L-DOPA (L-3,4-dihydroxyphenylalanine) were more potent inducers of apoptosis than proteins containing the phenylalanine oxidation product o-tyrosine. Apoptosis was dependent upon incorporation of oxidized residues, as indicated by complete abrogation in cultures incubated with the non-incorporation control D-DOPA (D-3,4-dihydroxyphenylalanine) or when incorporation was competed out by parent amino acids. The findings of the present study suggest that certain oxidized proteins could play an active role in the progression of age-related disorders by contributing to LMP (lysosomal membrane permeabilization)-initiated apoptosis and may have important implications for the long-term use of L-DOPA as a therapeutic agent in Parkinson's disease.
Enhancement of myosin II/actin turnover at the contractile ring induces slower furrowing in dividing HeLa cells
Authors: Kondo Tomo , Hamao Kozue , Kamijo Keiju , Kimura Hiroshi , Morita Makiko , Takahashi Masayuki , Hosoya Hiroshi
Published: February 2011

Myosin II ATPase activity is enhanced by the phosphorylation of MRLC (myosin II regulatory light chain) in non-muscle cells. It is well known that pMRLC (phosphorylated MRLC) co-localizes with F-actin (filamentous actin) in the CR (contractile ring) of dividing cells. Recently, we reported that HeLa cells expressing non-phosphorylatable MRLC show a delay in the speed of furrow ingression, suggesting that pMRLC plays an important role in the control of furrow ingression. However, it is still unclear how pMRLC regulates myosin II and F-actin at the CR to control furrow ingression during cytokinesis. In the present study, to clarify the roles of pMRLC, we measured the turnover of myosin II and actin at the CR in dividing HeLa cells expressing fluorescent-tagged MRLCs and actin by FRAP (fluorescence recovery after photobleaching). A myosin II inhibitor, blebbistatin, caused an enhancement of the turnover of MRLC and actin at the CR, which induced a delay in furrow ingression. Furthermore, only non-phosphorylatable MRLC and a Rho-kinase inhibitor, Y-27632, accelerated the turnover of MRLC and actin at the CR. Interestingly, the effect of Y-27632 was cancelled in the cell expressing phosphomimic MRLCs. Taken together, these results reveal that pMRLC reduces the turnover of myosin II and also actin at the CR. In conclusion, we show that the enhancement of myosin II and actin turnover at the CR induced slower furrowing in dividing HeLa cells.
Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape
Authors: Bartz René , Fan Haihong , Zhang Jingtao , Innocent Nathalie , Cherrin Craig , Beck Stephen C. , Pei Yi , Momose Aaron , Jadhav Vasant , Tellers David M. , Meng Fanyu , Crocker Louis S. , Sepp-Lorenzino Laura , Barnett Stanley F.
Published: February 2011

Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.
Light-induced blockage of cell division with a chromatin-targeted phototoxic fluorescent protein
Authors: Serebrovskaya Ekaterina O. , Gorodnicheva Tatiana V. , Ermakova Galina V. , Solovieva Elena A. , Sharonov George V. , Zagaynova Elena V. , Chudakov Dmitriy M. , Lukyanov Sergey , Zaraisky Andrey G. , Lukyanov Konstantin A.
Published: February 2011

Proteins of the GFP (green fluorescent protein) family are widely used as passive reporters for live cell imaging. In the present study we used H2B (histone H2B)–tKR (tandem KillerRed) as an active tool to affect cell division with light. We demonstrated that H2B–tKR-expressing cells behave normally in the dark, but transiently cease proliferation following green-light illumination. Complete light-induced blockage of cell division for approx. 24 h was observed in cultured mammalian cells that were either transiently or stably transfected with H2B–tKR. Illuminated cells then returned to normal division rate. XRCC1 (X-ray cross complementing factor 1) showed immediate redistribution in the illuminated nuclei of H2B–tKR-expressing cells, indicating massive light-induced damage of genomic DNA. Notably, nondisjunction of chromosomes was observed for cells that were illuminated during metaphase. In transgenic Xenopus embryos expressing H2B–tKR under the control of tissue-specific promoters, we observed clear retardation of the development of these tissues in green-light-illuminated tadpoles. We believe that H2B–tKR represents a novel optogenetic tool, which can be used to study mitosis and meiosis progression per se, as well as to investigate the roles of specific cell populations in development, regeneration and carcinogenesis in vivo.
C68 from the Sulfolobus islandicus plasmid–virus pSSVx is a novel member of the AbrB-like transcription factor family
Authors: Contursi Patrizia , D'Ambrosio Katia , Pirone Luciano , Pedone Emilia , Aucelli Tiziana , She Qunxin , De Simone Giuseppina , Bartolucci Simonetta
Published: February 2011

The genetic element pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. This plasmid–virus hybrid infects several species of the hyperthermophilic acidophilic crenarchaeon Sulfolobus. The open reading frame orfc68 of pSSVx encodes a 7.7 kDa protein that does not show significant sequence homology with any protein with known three-dimensional structure. EMSA (electrophoretic mobility-shift assay) experiments, DNA footprinting and CD analyses indicate that recombinant C68, purified from Escherichia coli, binds to two different operator sites that are located upstream of its own promoter. The three-dimensional structure, solved by a single-wavelength anomalous diffraction experiment on a selenomethionine derivative, shows that the protein assumes a swapped-hairpin fold, which is a distinctive fold associated with a family of prokaryotic transcription factors, such as AbrB from Bacillus subtilis. Nevertheless, C68 constitutes a novel representative of this family because it shows several peculiar structural and functional features.
Responses of hypertrophied myocytes to reactive species: implications for glycolysis and electrophile metabolism
Authors: Sansbury Brian E. , Riggs Daniel W. , Brainard Robert E. , Salabei Joshua K. , Jones Steven P. , Hill Bradford G.
Published: February 2011

During cardiac remodelling, the heart generates higher levels of reactive species; yet an intermediate ‘compensatory’ stage of hypertrophy is associated with a greater ability to withstand oxidative stress. The mechanisms underlying this protected myocardial phenotype are poorly understood. We examined how a cellular model of hypertrophy deals with electrophilic insults, such as would occur upon ischaemia or in the failing heart. For this, we measured energetics in control and PE (phenylephrine)-treated NRCMs (neonatal rat cardiomyocytes) under basal conditions and when stressed with HNE (4-hydroxynonenal). PE treatment caused hypertrophy as indicated by augmented atrial natriuretic peptide and increased cellular protein content. Hypertrophied myocytes demonstrated a 2.5-fold increase in ATP-linked oxygen consumption and a robust augmentation of oligomycin-stimulated glycolytic flux and lactate production. Hypertrophied myocytes displayed a protected phenotype that was resistant to HNE-induced cell death and a unique bioenergetic response characterized by a delayed and abrogated rate of oxygen consumption and a 2-fold increase in glycolysis upon HNE exposure. This augmentation of glycolytic flux was not due to increased glucose uptake, suggesting that electrophile stress results in utilization of intracellular glycogen stores to support the increased energy demand. Hypertrophied myocytes also had an increased propensity to oxidize HNE to 4-hydroxynonenoic acid and sustained less protein damage due to acute HNE insults. Inhibition of aldehyde dehydrogenase resulted in bioenergetic collapse when myocytes were challenged with HNE. The integration of electrophile metabolism with glycolytic and mitochondrial energy production appears to be important for maintaining myocyte homoeostasis under conditions of increased oxidative stress.
Changes in cytosolic Mg2+ levels can regulate the activity of the plasma membrane H+-ATPase in maize
Authors: Hanstein Stefan , Wang Xiaozhi , Qian Xiaoqing , Friedhoff Peter , Fatima Ammara , Shan Yuhua , Feng Ke , Schubert Sven
Published: February 2011

Plant PM (plasma membrane) H+-ATPase, a major consumer of cellular ATP, is driven by the MgATP complex which may dissociate at low cytosolic Mg2+ activity. We investigated whether hydrolytic activity of PM H+-ATPase is inhibited at ATP concentrations exceeding the Mg2+ concentration. Activity in isolated maize PMs was measured at pH 6.5 in the presence of 5 mM Mg2+ (high) or 2 mM Mg2+ (low), whereas K+ was applied at concentrations of 155 mM (high) or 55 mM (low). In all experiments, with membrane vesicles either from roots or leaves, the enzyme activity decreased in the presence of Mg2+-free ATP. At inhibitory ATP concentrations, the activity was not influenced by the K+ concentration. The activity was restored after increasing the Mg2+ concentration. ATP inhibition also occurred at pH 7.5. Kinetic modelling shows that Mg2+-free ATP acted as a competitive inhibitor with a Ki in the range of the Km. Ki decreased by 75% at low K+ concentration. Ki was one order of magnitude lower at pH 7.5 compared with pH 6.5. The observed inhibition is consistent with a concept in which down-regulation of the cytosolic Mg2+ activity is involved in (phyto)hormonal stress responses.
The role of TBK1 and IKKϵ in the expression and activation of Pellino 1
Authors: Smith Hilary , Liu Xin-Yu , Dai Liang , Goh Eddy T. H. , Chan Aye-Thu , Xi Jiajia , Seh Cheah-Chen , Qureshi Insaf A. , Lescar Julien , Ruedl Christiane , Gourlay Robert , Morton Simon , Hough Joanne , McIver Edward G. , Cohen Philip , Cheung Peter C. F.
Published: February 2011

Mammalian Pellino isoforms are phosphorylated by IRAK (interleukin receptor associated kinase) 1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. In the present paper we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDMs (bone-marrow-derived macrophages) are stimulated with LPS (lipopolysaccharide) or poly(I:C). This induction occurs via a TRIF [TIR (Toll/interleukin-1 receptor)-domain-containing adaptor-inducing interferon-β]-dependent IRAK-independent pathway and is prevented by inhibition of the IKK [IκB (inhibitor of nuclear factor κB) kinase]-related protein kinases, TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1} and IKKϵ. Pellino 1 is not induced in IRF3 (interferon regulatory factor 3)−/− BMDMs, and its induction is only reduced slightly in type 1 interferon receptor−/− BMDMs, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKϵ as bait, and show that IKKϵ/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288 and Ser293. Moreover, we show that the E3 ligase activity of endogenous Pellino 1 is activated in LPS- or poly(I:C)-stimulated macrophages. This occurs more rapidly than the increase in Pellino 1 mRNA and protein expression, is prevented by the inhibition of IKKϵ/TBK1 and is reversed by phosphatase treatment. Thus IKKϵ/TBK1 mediate the activation of Pellino 1's E3 ligase activity, as well as inducing the transcription of its gene and protein expression in response to TLR3 and TLR4 agonists.
Receptor-dependent compartmentalization of PPIP5K1, a kinase with a cryptic polyphosphoinositide binding domain
Authors: Gokhale Nikhil A. , Zaremba Angelika , Shears Stephen B.
Published: February 2011

The inositol pyrophosphates are multifunctional signalling molecules. One of the families of enzymes that synthesize the inositol pyrophosphates are the Vip1/PPIP5Ks (PP-InsP5 kinases). The kinase domains in Vip1/PPIP5Ks have been mapped to their N-terminus. Each of these proteins also possess a phosphatase-like domain of unknown significance. In the present study, we show that this phosphatase-like domain is not catalytically active. Instead, by using SPR (surface plasmon resonance) to study protein binding to immobilized lipid vesicles, we show that this domain is specialized for binding PtdIns(3,4,5)P3 (PPIP5K1 Kd=96 nM; PPIP5K2 Kd=705 nM). Both PtdIns(3,4)P2 and PtdIns(4,5)P2 are significantly weaker ligands, and no significant binding of PtdIns(3,5)P2 was detected. We confirm the functional importance of this domain in inositol lipid binding by site-directed mutagenesis. We present evidence that the PtdIns(3,4,5)P3-binding domain is an unusual hybrid, in which a partial PH (pleckstrin homology) consensus sequence is spliced into the phosphatase-like domain. Agonist-dependent activation of the PtdIns 3-kinase pathway in NIH 3T3 cells drives translocation of PPIP5K1 from the cytosol to the plasma membrane. We have therefore demonstrated receptor-regulated compartmentalization of inositol pyrophosphate synthesis in mammalian cells.
Crystal structure of Ssu72, an essential eukaryotic phosphatase specific for the C-terminal domain of RNA polymerase II, in complex with a transition state analogue
Authors: Zhang Yong , Zhang Mengmeng , Zhang Yan
Published: February 2011

Reversible phosphorylation of the CTD (C-terminal domain) of the eukaryotic RNA polymerase II largest subunit represents a critical regulatory mechanism during the transcription cycle and mRNA processing. Ssu72 is an essential phosphatase conserved in eukaryotes that dephosphorylates phosphorylated Ser5 of the CTD heptapeptide. Its function is implicated in transcription initiation, elongation and termination, as well as RNA processing. In the present paper we report the high resolution X-ray crystal structures of Drosophila melanogaster Ssu72 phosphatase in the apo form and in complex with an inhibitor mimicking the transition state of phosphoryl transfer. Ssu72 facilitates dephosphorylation of the substrate through a phosphoryl-enzyme intermediate, as visualized in the complex structure of Ssu72 with the oxo-anion compound inhibitor vanadate at a 2.35 Å (1 Å=0.1 nm) resolution. The structure resembles the transition state of the phosphoryl transfer with vanadate exhibiting a trigonal bi-pyramidal geometry covalently bonded to the nucleophilic cysteine residue. Interestingly, the incorporation of oxo-anion compounds greatly stabilizes a flexible loop containing the general acid, as detected by an increase of melting temperature of Ssu72 detected by differential scanning fluorimetry. The Ssu72 structure exhibits a core fold with a similar topology to that of LMWPTPs [low-molecular-mass PTPs (protein tyrosine phosphatases)], but with an insertion of a unique ‘cap’ domain to shelter the active site from the solvent with a deep groove in between where the CTD substrates bind. Mutagenesis studies in this groove established the functional roles of five residues (Met17, Pro46, Asp51, Tyr77 and Met85) that are essential specifically for substrate recognition.
AMP-activated protein kinase (AMPK) is a tau kinase, activated in response to amyloid β-peptide exposure
Authors: Thornton Claire , Bright Nicola J. , Sastre Magdalena , Muckett Phillip J. , Carling David
Published: February 2011

Hyperphosphorylation of tau is a hallmark of Alzheimer's disease and other tauopathies. Although the mechanisms underlying hyperphosphorylation are not fully understood, cellular stresses such as impaired energy metabolism are thought to influence the signalling cascade. The AMPK (AMP-activated protein kinase)-related kinases MARK (microtubule-associated protein-regulating kinase/microtubule affinity-regulating kinase) and BRSK (brain-specific kinase) have been implicated in tau phosphorylation, but are insensitive to activation by cellular stress. In the present study, we show that AMPK itself phosphorylates tau on a number of sites, including Ser262 and Ser396, altering microtubule binding of tau. In primary mouse cortical neurons, CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) activation of AMPK in response to Aβ (amyloid-β peptide)-(1–42) leads to increased phosphorylation of tau at Ser262/Ser356 and Ser396. Activation of AMPK by Aβ-(1–42) is inhibited by memantine, a partial antagonist of the NMDA (N-methyl-D-aspartate) receptor and currently licensed for the treatment of Alzheimer's disease. These findings identify a pathway in which Aβ-(1–42) activates CaMKKβ and AMPK via the NMDA receptor, suggesting the possibility that AMPK plays a role in the pathophysiological phosphorylation of tau.
Counter-modulation of fatty acid-induced pro-inflammatory nuclear factor κB signalling in rat skeletal muscle cells by AMP-activated protein kinase
Authors: Green Charlotte J. , Macrae Katherine , Fogarty Sarah , Hardie D. Grahame , Sakamoto Kei , Hundal Harinder S.
Published: February 2011

Sustained over-supply of saturated non-esterified ‘free’ fatty acids has been shown to promote skeletal muscle insulin resistance, which may be driven, in part, by an increase in inflammatory signalling within this tissue. In the present manuscript we show that exposure of L6 myotubes to palmitate, a saturated fatty acid, induces activation of the NF-κB (nuclear factor κB) pathway {based on increased IKK [IκB (inhibitory κB) kinase] phosphorylation, IκBα loss and elevated interleukin-6 mRNA expression} and that this was associated with enhanced phosphorylation/activation of p38 MAPK (mitogen-activated protein kinase), JNK (c-Jun N-terminal kinase) and ERK (extracellular-signal-regulated kinase) as well as impaired insulin-dependent activation of PKB (protein kinase B)/Akt and glucose transport. NF-κB activation by palmitate was unaffected by pharmacological inhibition of p38 MAPK or JNK, but was suppressed significantly by inhibition of MEK (MAPK/ERK kinase)/ERK signalling. The importance of ERK with respect to downstream NF-κB signalling was underscored by the finding that PMA, a potent ERK activator, enhanced IKK phosphorylation. Strikingly, both palmitate- and PMA-induced activation of IKK/NF-κB were antagonized by AMPK (AMP-activated protein kinase) activators because of reduced ERK signalling. Although palmitate-induced activation of NF-κB was repressed by AMPK activation and by cellular overexpression of a mutated IκBα (S32A/S36A) super-repressor, this did not ameliorate the loss in insulin-stimulated PKB activation or glucose transport. Our results from the present study indicate that ERK plays a pivotal role in palmitate-induced activation of the IKK/NF-κB signalling axis and that AMPK can restrain the activity of this pro-inflammatory pathway. The finding that insulin resistance persists in myotubes in which NF-κB signalling has been repressed implies that palmitate and/or its lipid derivatives retain the capacity to impair insulin-regulated events independently of the increase in inflammatory signalling.
Visualization of retrovirus uptake and delivery into acidic endosomes
Authors: Miyauchi Kosuke , Marin Mariana , Melikyan Gregory B.
Published: February 2011

Diverse enveloped viruses enter cells by endocytosis and fusion with intracellular compartments. Recent evidence suggests that HIV also infects permissive cell lines by fusing with endosomes in a pH-independent manner. This finding highlights the importance of time-resolved monitoring of viral uptake. In the present study, we designed an imaging-based assay to measure endocytosis in real-time through probing the virus' accessibility to external solutions. Exposure of viruses bearing a pH-sensitive GFP (green fluorescent protein) variant on their surface to solutions of different acidity altered the fluorescence of surface-accessible particles, but not internalized viruses. By sequentially applying acidic and alkaline buffers with or without ammonium chloride, we were able to quantify the fractions of internalized and non-internalized virions, as well as the fraction of detached particles, over time. The exact time of single-virus internalization was assessed from the point when a particle ceased to respond to a perfusion with alternating acidic and alkaline buffers. We found that, surprisingly, HIV pseudoparticles entered acidic compartments shortly after internalization. These results suggest that the virus might be sorted to a quickly maturing pool of endocytic vesicles and thus be trafficked to fusion-permissive sites near the cell nucleus.
How the biotin–streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer
Authors: Chivers Claire E. , Koner Apurba L. , Lowe Edward D. , Howarth Mark
Published: February 2011

The interaction between SA (streptavidin) and biotin is one of the strongest non-covalent interactions in Nature. SA is a widely used tool and a paradigm for protein–ligand interactions. We previously developed a SA mutant, termed Tr (traptavidin), possessing a 10-fold lower off-rate for biotin, with increased mechanical and thermal stability. In the present study, we determined the crystal structures of apo-Tr and biotin–Tr at 1.5 Å resolution. In apo-SA the loop (L3/4), near biotin's valeryl tail, is typically disordered and open, but closes upon biotin binding. In contrast, L3/4 was shut in both apo-Tr and biotin–Tr. The reduced flexibility of L3/4 and decreased conformational change on biotin binding provide an explanation for Tr's reduced biotin off- and on-rates. L3/4 includes Ser45, which forms a hydrogen bond to biotin consistently in Tr, but erratically in SA. Reduced breakage of the biotin–Ser45 hydrogen bond in Tr is likely to inhibit the initiating event in biotin's dissociation pathway. We generated a Tr with a single biotin-binding site rather than four, which showed a simi-larly low off-rate, demonstrating that Tr's low off-rate was governed by intrasubunit effects. Understanding the structural features of this tenacious interaction may assist the design of even stronger affinity tags and inhibitors.
Cold sensitivity in rice (Oryza sativa L.) is strongly correlated with a naturally occurring I99V mutation in the multifunctional glutathione transferase isoenzyme GSTZ2
Authors: Kim Sang-Ic , Andaya Virgilio C. , Tai Thomas H.
Published: February 2011

GSTZs [Zeta class GSTs (glutathione transferases)] are multifunctional enzymes that belong to a highly conserved subfamily of soluble GSTs found in species ranging from fungi and plants to animals. GSTZs are known to function as MAAIs [MAA (maleylacetoacetate) isomerases], which play a role in tyrosine catabolism by catalysing the isomerization of MAA to FAA (fumarylacetoacetate). As tyrosine metabolism in plants differs from animals, the significance of GSTZ/MAAI is unclear. In rice (Oryza sativa L.), a major QTL (quantitative trait locus) for seedling cold tolerance has been fine mapped to a region containing the genes OsGSTZ1 and OsGSTZ2. Sequencing of tolerant (ssp. japonica cv. M-202) and sensitive (ssp. indica cv. IR50) cultivars revealed two SNPs (single nucleotide polymorphisms) in OsGSTZ2 that result in amino acid differences (I99V and N184I). Recombinant OsGSTZ2 containing the Val99 residue found in IR50 had significantly reduced activity on MAA and DCA (dichloroacetic acid), but the Ile184 residue had no effect. The distribution of the SNP (c.295A>G) among various rice accessions indicates a significant association with chilling sensitivity in rice seedlings. The results of the present study show that naturally occurring OsGSTZ2 isoforms differ in their enzymatic properties, which may contribute to the differential response to chilling stress generally exhibited by the two major rice subspecies.
The progesterone receptor as a transcription factor regulates phospholipase D1 expression through independent activation of protein kinase A and Ras during 8-Br-cAMP-induced decidualization in human endometrial stromal cells
Authors: Cho Ju Hwan , Yoon Mee-Sup , Koo Jun Bon , Kim Yong Seok , Lee Ki-Sung , Lee Jung Han , Han Joong-Soo
Published: February 2011

Decidualization is a biological and morphological process occurring in hES (human endometrial stromal) cells. Previously, we reported that PLD1 (phospholipase D1) plays an important role in cAMP-induced decidualization of hES cells. In the present study, we focused on how PLD1 expression is up-regulated during decidualization. Treatment with PKA (protein kinase A) inhibitors (Rp-cAMP or H89) or a Ras inhibitor (manumycin) partially inhibited PLD1 expression and decidua formation in response to cAMP treatment. Interestingly, dual inhibition of PKA and Ras completely inhibited PLD1 expression and cAMP-induced decidualization. These results suggest that PLD1 expression during decidualization is controlled additively by PKA and Ras. The use of inhibitors showed that extracellular-signal-regulated kinase, a downstream effector of Ras, was required for PLD activation and the morphological changes during decidualization, but not for the increase in PLD1 protein. Next, to investigate the regulator of the PLD1 gene at the transcriptional level, a promoter assay using deletion mutants of the PLD1 promoter was performed; the result indicated that PR (progesterone receptor) was a possible regulator of the PLD1 gene. In addition, chromatin immunoprecipitation assays on the PLD1 promoter identified PR as a transcription factor for PLD1 expression during 8-Br-cAMP-induced decidualization. Taken together, our findings demonstrate that PKA and Ras are novel regulators of PLD1 expression and also identify PR as a transcription factor for PLD1 expression during the decidualization of hES cells.
X-ray structural studies of the entire extracellular region of the serine/threonine kinase PrkC from Staphylococcus aureus
Authors: Ruggiero Alessia , Squeglia Flavia , Marasco Daniela , Marchetti Roberta , Molinaro Antonio , Berisio Rita
Published: February 2011

Bacterial serine/threonine kinases modulate a wide number of cellular processes. The serine/threonine kinase PrkC from the human pathogen Staphylococcus aureus was also shown to induce germination of Bacillus subtilis spores, in response to cell wall muropeptides. The presence of muropeptides in the bacterial extracellular milieu is a strong signal that the growing conditions are promising. In the present paper, we report the X-ray structure of the entire extracellular region of PrkC from S. aureus. This structure reveals that the extracellular region of PrkC, EC-PrkC, is a linear modular structure composed of three PASTA (penicillin binding-associated and serine/threonine kinase-associated) domains and an unpredicted C-terminal domain, which presents the typical features of adhesive proteins. Using several solution techniques, we also found that EC-PrkC shows no tendency to dimerize even in the presence of high concentrations of muropeptides. X-ray structural results obtained in the present study provide molecular clues into the mechanism of muropeptide-induced PrkC activation.
Peroxisomal multifunctional enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics
Authors: Haataja Tatu J. K. , Koski M. Kristian , Hiltunen J. Kalervo , Glumoff Tuomo
Published: February 2011

All of the peroxisomal β-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type 2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure–function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2.
Spectroscopic and magnetic studies of wild-type and mutant forms of the Fe(II)- and 2-oxoglutarate-dependent decarboxylase ALKBH4
Authors: Bjørnstad Linn G. , Zoppellaro Giorgio , Tomter Ane B. , Falnes Pål Ø. , Andersson K. Kristoffer
Published: February 2011

The Fe(II)/2OG (2-oxoglutarate)-dependent dioxygenase superfamily comprises proteins that couple substrate oxidation to decarboxylation of 2OG to succinate. A member of this class of mononuclear non-haem Fe proteins is the Escherichia coli DNA/RNA repair enzyme AlkB. In the present work, we describe the magnetic and optical properties of the yet uncharacterized human ALKBH4 (AlkB homologue). Through EPR and UV–visible spectroscopy studies, we address the Fe-binding environment of the proposed catalytic centre of wild-type ALKBH4 and an Fe(II)-binding mutant. We could observe a novel unusual Fe(III) high-spin EPR-active species in the presence of sulfide with a gmax of 8.2. The Fe(II) site was probed with NO. An intact histidine-carboxylate site is necessary for productive Fe binding. We also report the presence of a unique cysteine-rich motif conserved in the N-terminus of ALKBH4 orthologues, and investigate its possible Fe-binding ability. Furthermore, we show that recombinant ALKBH4 mediates decarboxylation of 2OG in absence of primary substrate. This activity is dependent on Fe as well as on residues predicted to be involved in Fe(II) co-ordination. The present results demonstrate that ALKBH4 represents an active Fe(II)/2OG-dependent decarboxylase and suggest that the cysteine cluster is involved in processes other than Fe co-ordination.
WT1 and its transcriptional cofactor BASP1 redirect the differentiation pathway of an established blood cell line
Authors: Goodfellow Sarah J. , Rebello Michelle R. , Toska Eneda , Zeef Leo A. H. , Rudd Sean G. , Medler Kathryn F. , Roberts Stefan G. E.
Published: February 2011

The Wilms' tumour suppressor WT1 (Wilms' tumour 1) is a transcriptional regulator that plays a central role in organogenesis, and is mutated or aberrantly expressed in several childhood and adult malignancies. We previously identified BASP1 (brain acid-soluble protein 1) as a WT1 cofactor that suppresses the transcriptional activation function of WT1. In the present study we have analysed the dynamic between WT1 and BASP1 in the regulation of gene expression in myelogenous leukaemia K562 cells. Our findings reveal that BASP1 is a significant regulator of WT1 that is recruited to WT1-binding sites and suppresses WT1-mediated transcriptional activation at several WT1 target genes. We find that WT1 and BASP1 can divert the differentiation programme of K562 cells to a non-blood cell type following induction by the phorbol ester PMA. WT1 and BASP1 co-operate to induce the differentiation of K562 cells to a neuronal-like morphology that exhibits extensive arborization, and the expression of several genes involved in neurite outgrowth and synapse formation. Functional analysis revealed the relevance of the transcriptional reprogramming and morphological changes, in that the cells elicited a response to the neurotransmitter ATP. Taken together, the results of the present study reveal that WT1 and BASP1 can divert the lineage potential of an established blood cell line towards a cell with neuronal characteristics.
A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase
Authors: Gracia Eduard , Pérez-Capote Kamil , Moreno Estefanía , Barkešová Jana , Mallol Josefa , Lluís Carme , Franco Rafael , Cortés Antoni , Casadó Vicent , Canela Enric I.
Published: February 2011

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.
Crystal structure of unliganded TRAP: implications for dynamic allostery
Authors: Malay Ali D. , Watanabe Masahiro , Heddle Jonathan G. , Tame Jeremy R. H.
Published: February 2011

Allostery is vital to the function of many proteins. In some cases, rather than a direct steric effect, mutual modulation of ligand binding at spatially separated sites may be achieved through a change in protein dynamics. Thus changes in vibrational modes of the protein, rather than conformational changes, allow different ligand sites to communicate. Evidence for such an effect has been found in TRAP (trp RNA-binding attenuation protein), a regulatory protein found in species of Bacillus. TRAP is part of a feedback system to modulate expression of the trp operon, which carries genes involved in tryptophan synthesis. Negative feedback is thought to depend on binding of tryptophan-bound, but not unbound, TRAP to a specific mRNA leader sequence. We find that, contrary to expectations, at low temperatures TRAP is able to bind RNA in the absence of tryptophan, and that this effect is particularly strong in the case of Bacillus stearothermophilus TRAP. We have solved the crystal structure of this protein with no tryptophan bound, and find that much of the structure shows little deviation from the tryptophan-bound form. These data support the idea that tryptophan may exert its effect on RNA binding by TRAP through dynamic and not structural changes, and that tryptophan binding may be mimicked by low temperature.
SUMO E3 ligases are expressed in the retina and regulate SUMOylation of the metabotropic glutamate receptor 8b
Authors: Dütting Eva , Schröder-Kress Nadja , Sticht Heinrich , Enz Ralf
Published: February 2011

The central nervous system regulates neuronal excitability by macromolecular signalling complexes that consist of functionally related proteins, including neurotransmitter receptors, enzymes and scaffolds. The composition of these signal complexes is regulated by post-translational modifications, such as phosphorylation and SUMOylation (SUMO is small ubiquitin-related modifier). In the present study, we searched for proteins interacting with the intracellular C-termini of the metabotropic glutamate receptors mGluR8a and mGluR8b and identified proteins of the SUMOylation and NEDDylation machinery. The SUMO E3 ligases Pias1 [Pias is protein inhibitor of activated STAT (signal transducer and activator of transcription)] and Pias3L interacted strongly with mGluR8b, and were co-localized with the E2-conjugating Ubc9, SUMO1 and mGluR8b in cell bodies present in the ganglion cell layer of the mammalian retina. SUMO1 conjugation of Lys882, present in a bona fide consensus sequence for SUMOylation (VKSE) in the mGluR8b C-terminus, was enhanced by addition of Pias1, consistent with an interaction between both proteins. Mutation of Lys882 to arginine reduced, but did not abolish, mGluR8b SUMOylation. Co-mutating a second lysine residue (Lys903) located in the mGluR8b isoform-specific C-terminus largely prevented SUMO1 conjugation by Ubc9. Modelling studies suggested that Lys903 contacts Ubc9 and thus is part of the non-canonical SUMOylation site VKSG. In summary, the results of the present study show in vivo SUMOylation of the complete mGluR8b and co-localize proteins of the SUMOylation machinery in the retina.
Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH–cytochrome P450 reductase
Authors: Aigrain Louise , Pompon Denis , Truan Gilles
Published: February 2011

CPR (NADPH–cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the interfaces between the catalytic domains and the impact of interdomain interactions on the redox potentials of the flavins, the internal electron-transfer efficiency and the global conformation and dynamic equilibrium of the CPRs.
Galactose induction of the GAL1 gene requires conditional degradation of the Mig2 repressor
Authors: Lim Mei Kee , Siew Wee Leng , Zhao Jin , Tay Ywee Chieh , Ang Edwin , Lehming Norbert
Published: February 2011

Skp1 an essential component of the SCF (Skp1/cullin/F-box) E3 ubiquitin ligases, which target proteins for degradation by the 26S proteasome. We generated a skp1dM mutant strain that is defective for galactose induction of the GAL1 gene and we have found that galactose-induced protein degradation of the repressor Mig2 is defective in this strain. Mig2 degradation was also abolished in cells lacking the protein kinase Snf1 and the F-box protein Das1, suggesting that Snf1 triggers galactose-induced protein degradation of Mig2 by SCFDas1. Chromatin immunoprecipitation showed that Mig2 associates with the GAL1 promoter upon the galactose-induced exit of Mig1 in skp1dM cells, but not in wild-type cells, suggesting that the conditional degradation of Mig2 is required to prevent it from binding to the GAL1 promoter under inducing conditions. A galactose-stable deletion derivative of Mig2 caused a strong Mig (multi-copy inhibition of GAL gene expression) phenotype, confirming that galactose induction of the GAL1 gene requires the degradation of the repressor Mig2. Our results shed new light on the conflicting reports about the functional role of the degradation of transcriptional activators and indicate that gene expression studies interfering with proteasome degradation should take the stabilization of potential repressors into account.
Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides
Authors: Zervoudi Efthalia , Papakyriakou Athanasios , Georgiadou Dimitra , Evnouchidou Irini , Gajda Anna , Poreba Marcin , Salvesen Guy S. , Drag Marcin , Hattori Akira , Swevers Luc , Vourloumis Dionisios , Stratikos Efstratios
Published: February 2011

ERAP1 (endoplasmic reticulum aminopeptidase 1), ERAP2 and IRAP (insulin-regulated aminopeptidase) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding on to MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorigenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the S1 (primary specificity) pocket. Molecular modelling of the three S1 pockets reveals substrate–enzyme interactions that are critical determinants for specificity. The substrate selectivity profiles suggest that IRAP largely combines the S1 specificity of ERAP1 and ERAP2, consistent with its proposed biological function. IRAP, however, does not achieve this dual specificity by simply combining structural features of ERAP1 and ERAP2, but rather by an unique amino acid change at position 541. The results of the present study provide insights on antigenic peptide selection and may prove valuable in designing selective inhibitors or activity markers for this class of enzymes.
N-Docosahexaenoylethanolamide promotes development of hippocampal neurons
Authors: Kim Hee-Yong , Moon Hyun-Seuk , Cao Dehua , Lee Jeongrim , Kevala Karl , Jun Sang Beom , Lovinger David M. , Akbar Mohammed , Huang Bill X.
Published: February 2011

DHA (docosahexaenoic acid, C22:6,n−3) has been shown to promote neurite growth and synaptogenesis in embryonic hippocampal neurons, supporting the importance of DHA known for hippocampus-related learning and memory function. In the present study, we demonstrate that DHA metabolism to DEA (N-docosahexaenoylethanolamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. We found that a fatty acid amide hydrolase inhibitor URB597 potentiates DHA-induced neurite growth, synaptogenesis and synaptic protein expression. Active metabolism of DHA to DEA was observed in embryonic day 18 hippocampal neuronal cultures, which was increased further by URB597. Synthetic DEA promoted hippocampal neurite growth and synaptogenesis at substantially lower concentrations in comparison with DHA. DEA-treated neurons increased the expression of synapsins and glutamate receptor subunits and exhibited enhanced glutamatergic synaptic activity, as was the case for DHA. The DEA level in mouse fetal hippocampi was altered according to the maternal dietary supply of n–3 fatty acids, suggesting that DEA formation is a relevant in vivo process responding to the DHA status. In conclusion, DHA metabolism to DEA is a significant biochemical mechanism for neurite growth, synaptogenesis and synaptic protein expression, leading to enhanced glutamatergic synaptic function. The novel DEA-dependent mechanism offers a new molecular insight into hippocampal neurodevelopment and function.
IQ-motif selectivity in human IQGAP2 and IQGAP3: binding of calmodulin and myosin essential light chain
Authors: Atcheson Erwan , Hamilton Elaine , Pathmanathan Sevvel , Greer Brett , Harriott Pat , Timson David J.
Published: February 2011

The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain Mlc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed α-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins.