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  • 1. Aguiló, Francesca
    et al.
    Camarero, Nuria
    Relat, Joana
    Marrero, Pedro F
    Haro, Diego
    Transcriptional regulation of the human acetoacetyl-CoA synthetase gene by PPARgamma.2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 427, no 2Article in journal (Refereed)
    Abstract [en]

    In the cytosol of lipogenic tissue, ketone bodies are activated by AACS (acetoacetyl-CoA synthetase) and incorporated into cholesterol and fatty acids. AACS gene expression is particularly abundant in white adipose tissue, as it is induced during adipocyte differentiation. In order to elucidate the mechanism controlling the gene expression of human AACS and to clarify its physiological role, we isolated the human promoter, characterized the elements required to initiate transcription and analysed the expression of the gene in response to PPARgamma (peroxisome-proliferator-activated receptor gamma), an inducer of adipogenesis. We show that the human AACS promoter is a PPARgamma target gene and that this nuclear receptor is recruited to the AACS promoter by direct interaction with Sp1 (stimulating protein-1).

  • 2. Birkholtz, Lyn-Marie
    et al.
    Williams, Marni
    Niemand, Jandeli
    Louw, Abraham I.
    Persson, Lo
    Heby, Olle
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Polyamine homoeostasis as a drug target in pathogenic protozoa: peculiarities and possibilities2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 438, p. 229-244Article, review/survey (Refereed)
    Abstract [en]

    New drugs are urgently needed for the treatment of tropical and subtropical parasitic diseases, such as African sleeping sickness. Chagas' disease, leishmaniasis and malaria. Enzymes in polyamine biosynthesis and thiol metabolism, as well as polyamine transporters, are potential drug targets within these organisms. In the present review, the current knowledge of unique properties of polyamine metabolism in these parasites is outlined. These properties include prozyme regulation of AdoMetDC (S-adenosylmethionine decarboxylase) activity in trypanosomatids, co-expression of ODC (ornithine decarboxylase) and AdoMetDC activities in a single protein in plasmodia, and formation of trypanothione, a unique compound linking polyamine and thiol metabolism in trypanosomatids. Particularly interesting features within polyamine metabolism in these parasites are highlighted for their potential in selective therapeutic strategies.

  • 3.
    Birve, Simon
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Selstam, Eva
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Johansson, L B A
    Secondary structure of NADPH: protochlorophyllide oxidoreductase examined by circular dichroism and prediction methods1996In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 317, no 2, p. 549-555Article in journal (Refereed)
    Abstract [en]

    To study the secondary structure of the enzyme NADPH:protochlorophyllide oxidoreductase (PCOR), a novel method of enzyme isolation was developed. The detergent isotridecyl poly(ethylene glycol) ether (Genapol X-080) selectively solubilizes the enzyme from a prolamellar-body fraction isolated from wheat (Triticum aestivum L.). The solubilized fraction was further purified by ion-exchange chromatography. The isolated enzyme was studied by fluorescence spectroscopy at 77 K, and by CD spectroscopy. The fluorescence-emission spectra revealed that the binding properties of the substrate and co-substrate were preserved and that photo-reduction occurred. The CD spectra of PCOR were analysed for the relative amounts of the secondary structures, alpha-helix, beta-sheet, turn and random coil. The secondary structure composition was estimated to be 33% alpha-helix, 19% beta-sheet, 20% turn and 28% random coil. These values are in agreement with those predicted by the Predict Heidelberg Deutschland and self-optimized prediction method from alignments methods. The enzyme has some amino acid identity with other NADPH-binding enzymes containing the Rossmann fold. The Rossmann-fold fingerprint motif is localized in the N-terminal region and at the expected positions in the predicted secondary structure. It is suggested that PCOR is anchored to the interfacial region of the membrane by either a beta-sheet or an alpha-helical region containing tryptophan residues. A hydrophobic loop-region could also be involved in membrane anchoring.

  • 4.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Anders
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Lindhagen-Persson, Malin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ca2+ enhances Aβ polymerization rate and fibrillar stability in a dynamic manner2013In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 450, p. 189-197Article in journal (Refereed)
    Abstract [en]

    Identifying factors that affect the self-assembly of the amyloid-β peptide (Aβ) is of utmost importance in the quest to understand the molecular mechanisms causing Alzheimer's disease (AD). Ca2+ has previously been shown to accelerate both Aβ fibril nucleation and maturation, and a dysregulated Ca2+ homeostasis frequently correlates with development of AD. The mechanisms regarding Ca2+ binding as well as its effect on fibril kinetics are not fully understood. Using a polymerization assay we show that Ca2+ in a dynamic and reversible manner enhances both the elongation rate and fibrillar stability, where specifically the "dock and lock" phase mechanism is enhanced. Through NMR analysis we found that Ca2+ affects the fibrillar architecture. In addition, and unexpectedly, we found that Ca2+ does not bind the free Aβ monomer. This implies that Ca2+ binding requires an architecture adopted by assembled peptides, and consequently is mediated through intermolecular interactions between adjacent peptides. This gives a mechanistic explanation to the enhancing effect on fibril maturation and indicates structural similarities between prefibrillar structures and mature amyloid. Taken together we expose how Ca2+ levels affect the delicate equilibrium between the monomeric and assembled Aβ and how fluctuations in vivo may contribute to development and progression of the disease.

  • 5.
    Chevreuil, O
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Hultin, M
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Ostergaard, P
    Olivecrona, T
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Heparin-decasaccharides impair the catabolism of chylomicrons.1996In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 320 ( Pt 2), p. 437-44Article in journal (Refereed)
    Abstract [en]

    On intravenous injection to rats, decasaccharides gave rise to a short-lived peak of lipoprotein lipase (LPL) activity, whereas octa- and hexasaccharides caused only marginal increases. In isolated hearts perfused by a single pass, decasaccharides released LPL more efficiently than conventional heparin on a mass basis. Octa- and hexasaccharides were much less efficient. Similar results were obtained for hepatic lipase, which was studied both in vivo and by liver perfusion. In the intact rat, the heparin fragments themselves disappeared rapidly from the circulating blood. The decay of hepatic lipase activity after the early peak roughly paralleled the decay of decasaccharide concentration, but for LPL the decay was faster, presumably because the liver extracted this lipase from plasma. To assess the lipase activities remaining in contact with blood a large dose of conventional heparin was injected at a series of times after the decasaccharides. LPL was decreased by 40% after 1 h. At that time, the LPL activity that could be released from isolated hearts by single-pass perfusion with heparin for 2 min ("functional LPL') was decreased by 75%. Chylomicrons labelled in vivo with [14C]oleic acid (primarily in triacylglycerols, providing a tracer for lipolysis) and [3H]retinol (primarily in ester form, providing a tracer for the particles) were injected intravenously to explore the effects of the LPL depletion on lipoprotein metabolism. Triacylglycerol lipolysis and particle clearance was markedly delayed from 30 min to 2 h after injection of decasaccharides. After 1 h the fractional catabolic rate was only one-third of the control value and the catabolism of chylomicron triacylglycerols by perfused hearts was delayed to a similar extent. Thus injection of decasaccharides leads to accelerated turnover of LPL with loss of functional LPL from extrahepatic tissues. This in turn leads to a period of delayed lipolysis and removal of chylomicron particles.

  • 6. de Veer, Simon J.
    et al.
    Swedberg, Joakim E.
    Akcan, Muharrem
    Rosengren, K. Johan
    Brattsand, Maria
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Craik, David J.
    Harris, Jonathan M.
    Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition2015In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 469, no 2, p. 243-253Article in journal (Refereed)
    Abstract [en]

    Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.

  • 7. Dejardin, A
    et al.
    Sokolov, L N
    Kleczkowski, Leszek A
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Sugar/osmoticum levels modulate differential abscisic acid-independent expression of two stress-responsive sucrose synthase genes in Arabidopsis1999In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 344, p. 503-509Article in journal (Refereed)
    Abstract [en]

    Sucrose synthase (Sus) is a key enzyme of sucrose metabolism. Two Sus-encoding genes (Sus1 and Sus2) from Arabidopsis thaliana were found to be profoundly and differentially regulated in leaves exposed to environmental stresses (cold stress, drought or O-2 deficiency). Transcript levels of Sus1 increased on exposure to cold and drought, whereas Sus2 mRNA was induced specifically by O-2 deficiency. Both cold and drought exposures induced the accumulation of soluble sugars and caused a decrease in leaf osmotic potential, whereas O-2 deficiency was characterized by a nearly complete depletion in sugars. Feeding abscisic acid (ABA) to detached leaves or subjecting Arabidopsis ABA-deficient mutants to cold stress conditions had no effect on the expression profiles of Sus1 or Sus2, whereas feeding metabolizable sugars (sucrose or glucose) or non-metabolizable osmotica [poly(ethylene glycol), sorbitol or mannitol] mimicked the effects of osmotic stress on Sus1 expression in detached leaves. By using various sucrose/mannitol solutions, we demonstrated that Sus1 was up-regulated by a decrease in leaf osmotic potential rather than an increase in sucrose concentration itself. We suggest that Sus1 expression is regulated via an ABA-independent signal transduction pathway that is related to the perception of a decrease in leaf osmotic potential during stresses. In contrast, the expression of Sus2 was independent of sugar/osmoticum effects, suggesting the involvement of a signal transduction mechanism distinct from that regulating Sus1 expression. The differential stress-responsive regulation of Sus genes in leaves might represent part of a general cellular response to the allocation of carbohydrates during acclimation processes.

  • 8. Eberth, Alexander
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gremer, Lothar
    Dvorsky, Radovan
    Koessmeier, Katja T
    McMahon, Harvey T
    Ahmadian, Mohammad Reza
    A BAR domain-mediated autoinhibitory mechanism for RhoGAPs of the GRAF family2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 417, no 1, p. 371-377Article in journal (Refereed)
    Abstract [en]

    The BAR (Bin/amphiphysin/Rvs) domain defines an emerging superfamily of proteins implicated in fundamental biological processes by sensing and inducing membrane curvature. We identified a novel autoregulatory function for the BAR domain of two related GAPs' (GTPase-activating proteins) of the GRAF (GTPase regulator associated with focal adhesion kinase) subfamily. We demonstrate that the N-terminal fragment of these GAPs including the BAR domain interacts directly with the GAP domain and inhibits its activity. Analysis of various BAR and GAP domains revealed that the BAR domain-mediated inhibition of these GAPs' function is highly specific. These GAPs, in their autoinhibited state, are able to bind and tubulate liposomes in vitro, and to generate lipid tubules in cells. Taken together, we identified BAR domains as cis-acting inhibitory elements that very likely mask the active sites of the GAP domains and thus prevent down-regulation of Rho proteins. Most remarkably, these BAR proteins represent a dual-site system with separate membrane-tubulation and GAP-inhibitory functions that operate simultaneously.

  • 9. Elle, Ida C
    et al.
    Simonsen, Karina T
    Olsen, Louise C B
    Birck, Pernille K
    Ehmsen, Sidse
    Tuck, Simon
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Le, Thuc T
    Færgeman, Nils J
    Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 437, no 2, p. 231-241Article in journal (Refereed)
    Abstract [en]

    ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the β-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased β-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.

  • 10.
    Henriksson, M L
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Telepnev, Maxim
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras2000In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 347, no 1, p. 217-222Article in journal (Refereed)
  • 11.
    Henriksson, Maria L.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sundin, Charlotta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Jansson, Anna L.
    Forsberg, Åke
    Palmer, Ruth H.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hallberg, Bengt
    Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo2002In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 367, no 3, p. 617-28Article in journal (Refereed)
    Abstract [en]

    Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells. ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities. We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo. In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo. We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P. aeruginosa. Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1.

  • 12.
    Henriksson, Maria L.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Trollér, Ulrika
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    14-3-3 proteins are required for the inhibition fo Ras by exoenzyme S2000In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 349, p. 697-701Article in journal (Refereed)
    Abstract [en]

    14-3-3 proteins play a regulatory role and participate in both signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine ligands, such as Raf-l kinase and Bad, by recognizing the phosphorylated consensus motif, Arg-Ser-Xaa-pSer-Xaa-Pro (where 'Xaa' represents 'any residue', and 'pSer' is 'phosphoserine'). However, 14-3-3 proteins must bind unphosphorylated ligands, such as glycoprotein Ib alpha and Pseudomonas aeruginosa exoenzyme S (ExoS), since it has been suggested that specific residues of 14-3-3 proteins are required for activation of ExoS. Furthermore, an unphosphorylated peptide derived from a phage display library inhibited the binding of both ExoS and Raf-1 to 14-3-3, and bound within the same conserved amphipathic groove on the surface of 14-3-3 as the Raf-derived phosphopeptide (pS-Raf-259). In the present study we identify the interaction site on ExoS for 14-3-3, and show that ExoS and 14-3-3 do indeed interact in vivo. In addition, we show that this interaction is critical for the ADP-ribosylation of Ras by ExoS, both in vitro and in vivo. Loss of the 14-3-3 binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras, and displays reduced killing activity.

  • 13.
    Huesgen, Pitter Florian
    et al.
    Department of Biology, University of Konstanz, Germany.
    Miranda, Helder
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lam, Xuan Tam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Perthold, Manuela
    Department of Biology, University of Konstanz, Germany.
    Schuhmann, Holger
    Department of Biology, University of Konstanz, Germany.
    Adamska, Iwona
    Department of Biology, University of Konstanz, Germany.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 435, no 3, p. 733-742Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria require efficient protein quality control mechanisms to survive under dynamic, often stressful environmental conditions. It was reported that three serine proteases, HtrA, HhoA and HhoB are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. Here we show that all three proteases can degrade unfolded model substrates, but differ in respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison to each other and to the well-studied Escherichia coli orthologues DegP and DegS. Deletion of the PDZ domain decreased, but not abolished proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterisation of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant stress resistance functions in Synechocystis sp. PCC 6803.

  • 14.
    Hultin, M
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Olivecrona, G
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Olivecrona, T
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Effect of protamine on lipoprotein lipase and hepatic lipase in rats.1994In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 304 ( Pt 3), p. 959-66Article in journal (Refereed)
    Abstract [en]

    The polycation protamine impedes the catabolism of triglyceride-rich lipoproteins and this has been suggested to be due to intravascular inactivation of lipoprotein lipase. We have made intravenous injections of protamine to rats and found that both lipoprotein lipase and hepatic lipase activities were released to plasma. The effect of protamine was more short-lived than that obtained by injection of heparin. The release of hepatic lipase by protamine was as effective as the release by heparin, while the amount of lipoprotein lipase released by protamine was only about one-tenth of that released by heparin. This was not due to inactivation of lipoprotein lipase, since injection of an excess of heparin 10 min after injection of protamine released as much lipoprotein lipase activity to plasma as in controls. The results in vivo differed from those obtained in model experiments in vitro. Protamine was able to almost quantitatively release both lipoprotein lipase and hepatic lipase from columns of heparin-agarose. The displacement was dependent on the total amount of protamine that had passed over the column, indicating that it was due to occupation by protamine of all available binding sites. Our results in vivo showed that the binding sites for lipoprotein lipase were not blocked as efficiently as those for hepatic lipase, indicating that the binding structures were not identical. It was concluded that the impaired turnover of lipoproteins by protamine probably was due to prevention of binding of the lipoproteins to endothelial cell surfaces rather than to impaired lipase function.

  • 15. Igamberdiev, A U
    et al.
    Kleczkowski, Leszek A
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments2001In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 360, p. 225-231Article in journal (Refereed)
    Abstract [en]

    On the basis of the equilibrium of adenylate kinase (AK; EC 2.7.4.3). which interconverts MgATP and free AMP with MgADP and free ADP, an approach has been worked out to calculate concentrations of free magnesium (Mg2+), based on concentrations of total ATP, ADP and AMP in plant tissues and in individual subcellular compartments. Based on reported total adenylate contents, [Mg2+] in plant tissues and organelles varies significantly depending on light and dark regimes, plant age and developmental stage. In steady-state conditions, [Mg2+] in chloroplasts is similar in light and darkness (in the millimolar range), whereas in the cytosol it is very low in the light and increases to about 0.4 mM in darkness. During the dark-to-light transition (photosynthetic induction), the [Mg2+] in chloroplasts falls to low values (0.2 mM or less), corresponding to a delay in photosynthetic oxygen evolution. This delay is considered to result from lower activities of Mg-dependent enzymes in the Calvin cycle. In mitochondria, the changes in [Mg2+] are similar but smoother. On the other hand, when the transition from light to darkness is considered, an initial increase in [Mg2+] occurs in both chloroplasts and mitochondria, which may be of importance for the control of key regulatory enzymes (e.g. mitochondrial malic enzyme and pyruvate dehydrogenase complex) and for processes connected with light-enhanced dark respiration. A rationale is presented for a possible role of [MgATP]/[MgADP] ratio (rather than [ATP(total)]/[ADP(total)]) as an important component of metabolic cellular control. It is postulated that assays of total adenylates may provide an accurate measure of [Mg2+] in plant tissues/cells and subcellular compartments, given that the adenylates are equilibrated by AK.

  • 16. Igamberdiev, Abir U
    et al.
    Kleczkowski, Leszek A
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Magnesium and cell energetics in plants under anoxia2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 437, no 3, p. 373-9Article in journal (Refereed)
    Abstract [en]

    Stress conditions (e.g. anoxia) frequently result in a decrease of [ATP] and in an increase of [ADP] and [AMP], with a concomitant increase of [Mg(2+)] and other cations, e.g. Ca(2+). The elevation of [Mg(2+)] is linked to the shift in the apparent equilibrium of adenylate kinase. As a result, enzymes that use Mg(2+) as a cofactor are activated, Ca(2+) activates calcium-dependent signalling pathways, and PP(i) can serve as an alternative energy source in its active form of MgPP(i) or Mg2PP(i). Under anoxic conditions in plants, an important source of PP(i) may come as a result of combined reactions of PK (pyruvate kinase) and PPDK (pyruvate, phosphate dikinase). The PP(i) formed in the PPDK/PK cycle ignites glycolysis in conditions of low [ATP] by involving PP(i)-dependent reactions. This saves ATP and makes metabolism under stress conditions more energy efficient.

  • 17.
    Kleczkowski, Leszek A
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Geisler, Matt
    Fitzek, Elisabeth
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases.2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 439, no 3, p. 375-379Article in journal (Refereed)
    Abstract [en]

    Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

  • 18.
    Kleczkowski, Leszek A.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Geisler, Matt
    Fitzek, Elisabeth
    Wilczynska, Malgorzata
    A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 439, p. 375-379Article, review/survey (Refereed)
    Abstract [en]

    Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

  • 19.
    Lammi, Mikko
    et al.
    Department of Anatomy, University of Kuopio, Kuopio,Finland.
    Inkinen, Ritva
    Department of Anatomy, University of Kuopio, Kuopio,Finland.
    Parkkinen, Jyrki
    Department of Pathology, University of Kuopio, Kuopio, Finland.
    Häkkinen, Tomi
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Jortikka, Matti
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Nelimarkka, Lassi
    Department of Medical Biochemistry, University of Turku, Turku, Finland.
    Järveläinen, Hannu
    Department of Medical Biochemistry, University of Turku, Turku, Finland.
    Tammi, Markku
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Expression of reduced amounts of structurally altered aggrecan in articular cartilage chondrocytes exposed to high hydrostatic pressure.1994In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 304, p. 723-730, article id 7818473Article in journal (Refereed)
    Abstract [en]

    The effect of hydrostatic pressure on proteoglycan (PG) metabolism of chondrocyte cultures was examined using a specially designed test chamber. Primary cultures of bovine articular chondrocytes at confluence were exposed for 20 h to 5 and 30 MPa continuous hydrostatic pressures and 5 MPa hydrostatic pulses (0.017, 0.25 and 0.5 Hz) in the presence of [35S]sulphate. Northern blot analyses showed that chondrocyte cultures used in this study expressed abundant mRNA transcripts of aggrecan, typical of chondrocytes, but not versican. The cultures also expressed biglycan and decorin. Enzymic digestions with keratanase and chondroitinases AC, ABC and B and subsequent SDS/agarose gel electrophoresis confirmed the synthesis of aggrecans and small dermatan sulphate PGs. The continuous 30 MPa pressure reduced total PG synthesis by 37% as measured by [35S]sulphate incorporation, in contrast to the 5 MPa continuous pressure which had no effect. The high static pressure also reduced total [3H]glucosamine incorporation by 63% and total [14C]leucine incorporation by 57%. The cyclic pressures showed a frequency-dependent stimulation (0.5 Hz, 11%) or inhibition (0.017 Hz, -17%) of [35S]sulphate incorporation. Aggrecans secreted under continuous 30 MPa pressure showed a retarded migration in 0.75% SDS/agarose gel electrophoresis and they also eluted earlier on Sephacryl S-1000 gel filtration, indicative of a larger molecular size. The increased size was consistent with an increase of average glycosaminoglycan chain length as determined by Sephacryl S-300 gel filtration. No change in aggrecan size was observed with the lower (5 MPa) static or cyclic pressures. Continuous 30 MPa hydrostatic pressure slightly reduced the steady-state mRNA level of aggrecan, in parallel with the decline in PG synthesis measured by [35S]sulphate incorporation. The results demonstrated that high hydrostatic pressure could influence the synthesis of PGs, especially of aggrecans, in chondrocytes both at the transcriptional and translational/post-translational levels.

  • 20.
    Liu, G
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Hultin, M
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Ostergaard, P
    Olivecrona, T
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Interaction of size-fractionated heparins with lipoprotein lipase and hepatic lipase in the rat.1992In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 285 ( Pt 3), p. 731-6Article in journal (Refereed)
    Abstract [en]

    Heparin and heparin partially depolymerized by enzymic digestion were separated into six size fractions. Hep 1 (tetrasaccharides), with a mean M(r) of 1200, did not release significant amounts of either lipoprotein lipase (LPL) or hepatic lipase (HL) on intravenous injection into rats. Hep 2 (mainly octa- and deca-saccharides), with a mean M(r) of 2400-3000, released both lipases. To evoke the same plasma activity of LPL and HL required about 10 times more by weight, or about 40 times more molecules, of this heparin than of hep 5 (mean M(r) 12,000, similar to conventional heparin). Hep 5 impeded binding and degradation of 125I-labelled bovine LPL by perfused rat livers. In contrast, hep 2 had no detectable effect on these processes. This demonstrates a difference between the sites in the liver that mediate binding, uptake and degradation of LPL, and the extrahepatic sites that bind functional LPL, and the hepatic sites that bind functional HL. After injection of 3.25 mg of hep 5/kg body weight, plasma LPL activity rapidly rose and then remained high for at least 1 h. With hep 2, plasma LPL also rose rapidly, but then decreased to almost basal by 1 h. When a labelled triacylglycerol emulsion was injected 1 h after the heparins, the fractional catabolic rate was enhanced in the rats that had received conventional heparin, as expected from the high plasma LPL activity, but decreased compared with controls in rats that had received hep 2, indicating that available LPL had been depleted through enhanced transport to and uptake in the liver.

  • 21.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The beta-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-22002In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 362, no 3, p. 597-607Article in journal (Refereed)
    Abstract [en]

    Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the beta-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the beta-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the beta-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel beta-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane.

  • 22.
    Marino, Giada
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Huesgen, Pitter F
    Eckhard, Ulrich
    Overall, Christopher M
    Schröder, Wolfgang P
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Family-wide characterization of Matrix Metallo-proteinases from Arabidopsis thaliana reveals their distinct proteolytic activity and cleavage site specificity.2014In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 457, no 2, p. 335-346Article in journal (Refereed)
    Abstract [en]

    Matrix metalloproteases (MMPs) are a family of zinc-dependent endopeptidases widely distributed throughout all kingdoms of life. In mammals, MMPs play key roles in many physiological and pathological processes including remodeling of the extracellular matrix. In the genome of the annual plant Arabidopsis thaliana five MMP-like proteins (At-MMPs) are encoded, but their function is unknown. Previous work on these enzymes was limited to gene expression analysis, and so far proteolytic activity has been shown only for At1-MMP. We expressed and purified the catalytic domains of all five At-MMPs as His-tagged proteins in E.coli to delineate the biochemical differences and similarities among the Arabidopsis MMP family members. We demonstrate that all five recombinant At-MMPs are active proteases with distinct preferences for different protease substrates. Furthermore, we performed a family-wide characterization of their biochemical properties and highlight similarities and differences in their cleavage site specificities as well as pH- and temperature dependent activities. Detailed analysis of their sequence specificity using Proteomic Identification of protease Cleavage Sites (PICS) revealed profiles similar to human MMPs with the exception of At5-MMP; homology models of the At-MMP catalytic domains supported these results. Our results suggest that each At-MMP may be involved in different proteolytic processes during plant growth and development.

  • 23.
    Martz, Françoise
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Wilczynska, Malgorzata
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Kleczkowski, Leszek A
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Oligomerization status, with the monomer as active species, defines catalytic efficiency of UDP-glucose pyrophosphorylase2002In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 367, no 1, p. 295-300Article in journal (Refereed)
    Abstract [en]

    Barley UDP-glucose pyrophosphorylase (UGPase), a key enzyme for the synthesis of sucrose, cellulose and other saccharides, was expressed in Escherichia coli and purified. Using both native electrophoresis and gel filtration, the recombinant and crude leaf UGPase proteins were found to exist as a mixture of monomers, dimers and higher-order polymers. In order to understand the molecular basis for the oligomerization of UGPase, a conserved Cys residue was replaced (C99S mutant) and several amino acids were substituted (LIV to NIN, KK to LL and LLL to NNN) in a conserved hydrophobic domain (amino acids 117-138). The C99S mutant had about half the V (max) of the wild-type and a 12-fold higher K (m) for PP(i), whereas NIN and LL mutations lowered the V (max) by 12- and 2-fold, respectively, with relatively small effects on substrate K (m) values (the NNN mutant was insoluble/inactive). The NIN mutation resulted in a low-activity oligomerized enzyme form, with very little monomer formation. Activity staining on native PAGE gels as well as gel-filtration studies demonstrated that the monomer was the sole enzymically active form. Possible implications of the oligomerization status of UGPase for post-translational regulation of the enzyme are discussed.

  • 24.
    Obi, Ikenna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Varying dependency of periplasmic peptidylprolyl cis-trans isomerases in promoting Yersinia pseudotuberculosis stress tolerance and pathogenicity2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 439, no 2, p. 321-332Article in journal (Refereed)
    Abstract [en]

    Periplasmic PPIases (peptidylprolyl cis-trans isomerases) catalyse the cis-trans isomerization of peptidyl-prolyl bonds, which is a rate-limiting step during protein folding. We demonstrate that the surA, ppiA, ppiD, fkpA and fklB alleles each encode a periplasmic PPIase in the bacterial pathogen Yersinia pseudotuberculosis. Of these, four were purified to homogeneity. Purified SurA, FkpA and FklB, but not PpiD, displayed detectable PPIase activity in vitro. Significantly, only Y. pseudotuberculosis lacking surA caused drastic alterations to the outer membrane protein profile and FA (fatty acid) composition. They also exhibited aberrant cellular morphology, leaking LPS (lipopolysaccharide) into the extracellular environment. The SurA PPIase is therefore most critical for maintaining Y. pseudotuberculosis envelope integrity during routine culturing. On the other hand, bacteria lacking either surA or all of the genes ppiA, ppiD, fkpA and fklB were sensitive to hydrogen peroxide and were attenuated in mice infections. Thus Y. pseudotuberculosis exhibits both SurA-dependent and -independent requirements for periplasmic PPIase activity to ensure in vivo survival and a full virulence effect in a mammalian host.

  • 25.
    Palmer, Ruth H
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Vernersson, Emma
    Grabbe, Caroline
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Anaplastic lymphoma kinase: signalling in development and disease.2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 420, no 3, p. 345-361Article in journal (Refereed)
    Abstract [en]

    RTKs (receptor tyrosine kinases) play important roles in cellular proliferation and differentiation. In addition, RTKs reveal oncogenic potential when their kinase activities are constitutively enhanced by point mutation, amplification or rearrangement of the corresponding genes. The ALK (anaplastic lymphoma kinase) RTK was originally identified as a member of the insulin receptor subfamily of RTKs that acquires transforming capability when truncated and fused to NPM (nucleophosmin) in the t(2;5) chromosomal rearrangement associated with ALCL (anaplastic large cell lymphoma). To date, many chromosomal rearrangements leading to enhanced ALK activity have been described and are implicated in a number of cancer types. Recent reports of the EML4 (echinoderm microtubule-associated protein like 4)-ALK oncoprotein in NSCLC (non-small cell lung cancer), together with the identification of activating point mutations in neuroblastoma, have highlighted ALK as a significant player and target for drug development in cancer. In the present review we address the role of ALK in development and disease and discuss implications for the future.

  • 26.
    Schönherr, Christina
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ruuth, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Yamazaki, Yasuo
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Eriksson, Therese
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Christensen, James
    Pfizer Global Research and Development, Department of Research Pharmacology, La Jolla Laboratories, La Jolla, CA 92121, U.S.A..
    Palmer, Ruth H
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE6842011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 440, p. 405-413Article in journal (Refereed)
    Abstract [en]

    Mutations in the kinase domain of ALK (anaplastic lymphoma kinase) have recently been shown to be important for the progression of the childhood tumour neuroblastoma. In the present study we investigate six of the putative reported constitutively active ALK mutations, in positions G1128A, I1171N, F1174L, R1192P, F1245C and R1275Q. Our analyses were performed in cell-culture-based systems with both mouse and human ALK mutant variants and subsequently in a Drosophila melanogaster model system. Our investigation addressed the transforming potential of the putative gain-of-function ALK mutations as well as their signalling potential and the ability of two ATP-competitive inhibitors, Crizotinib (PF-02341066) and NVP-TAE684, to abrogate the activity of ALK. The results of the present study indicate that all mutations tested are of an activating nature and thus are implicated in tumour initiation or progression of neuroblastoma. Importantly for neuroblastoma patients, all ALK mutations used in the present study can be blocked by the inhibitors, although some mutants exhibited higher levels of drug sensitivity than others.

  • 27. Shaikhali, Jehad
    et al.
    Davoine, Celine
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Rouhier, Nicolas
    Bygdell, Joakim
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Wingsle, Gunnar
    Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein2015In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 468, no 3, p. 385-400Article in journal (Refereed)
    Abstract [en]

    The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combinationwith specific TFs as GeBPL.

  • 28. Sokolov, L N
    et al.
    Dejardin, A
    Kleczkowski, Leszek A
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Sugars and light/dark exposure trigger differential regulation of ADP-glucose pyrophosphorylase genes in Arabidopsis thaliana (thale cress)1998In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 336, p. 681-687Article in journal (Refereed)
    Abstract [en]

    Expression of four Arabidopsis (thale cress) genes corresponding to the small (ApS) and large subunits (ApL1, ApL2, ApL3) of ADP-glucose pyrophosphorylase (AGPase), a key enzyme of starch biosynthesis, was found to be profoundly and differentially regulated by sugar and light/dark exposures. Transcript levels of both ApL2 and ApL3, and to a lesser extent ApS, increased severalfold upon feeding sucrose or glucose to the detached leaves in the dark, whereas the mRNA content for ApL1 decreased under the same conditions. Glucose was, in general, less effective than sucrose in inducing regulation of AGPase genes, possibly due to observed limitations in its uptake when compared with sucrose uptake by detached leaves. Osmotic agents [sorbitol, poly(ethylene glycol)] had no effect on ApS, ApL2 and ApL3 transcript level, but they did mimic the effect of sucrose on ApL1 gene, suggesting that the latter is regulated by osmotic pressure rather than any particular sugar. For all the genes the sugar effect was closely mimicked by an exposure of the dark-pre-adapted leaves to the light. Under both dark and light conditions, sucrose fed to the detached leaves was found to be rapidly metabolized to hexoses and, to some extent, starch. Starch production reflected most probably an increase in substrate availability for AGPase reaction rather than being due to changes in AGPase protein content, since both the sugar feeding and light exposure had little or no effect on the activity of AGPase or on the levels of its small and large subunit proteins in leaf extracts. The data suggest tight translational or posttranslational control, but they may also reflect spatial control of AGPase gene expression within a leaf. The sugar/light-dependent regulation of AGPase gene expression may represent a part of a general cellular response to the availability/allocation of carbohydrates during photosynthesis.

  • 29.
    Tam, Lam Xuan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Aigner, Raik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Timmerman, E
    Gevaert, K
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Proteomic approaches to identify substrates of the three Deg/HtrA proteases of the cyanobacterium Synechocystis sp PCC 68032015In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 468, no 3, p. 373-384Article in journal (Refereed)
    Abstract [en]

    The family of Deg/HtrA proteases plays an important role in quality control of cellular proteins in a wide range of organisms. In the genome of the cyanobacterium Synechocystis sp. PCC 6803, a model organism for photosynthetic research and renewable energy products, three Deg proteases are encoded, termed HhoA, HhoB and HtrA. In the present study, we compared wild-type (WT) Synechocystis cells with the single insertion mutants ΔhhoA, ΔhhoB and ΔhtrA. Protein expression of the remaining Deg/HtrA proteases was strongly affected in the single insertion mutants. Detailed proteomic studies using DIGE (difference gel electrophoresis) and N-terminal COFRADIC (N-terminal combined fractional diagonal chromatography) revealed that inactivation of a single Deg protease has similar impact on the proteomes of the three mutants; differences to WT were observed in enzymes involved in the major metabolic pathways. Changes in the amount of phosphate permease system Pst-1 were observed only in the insertion mutant ΔhhoB. N-terminal COFRADIC analyses on cell lysates of ΔhhoB confirmed changed amounts of many cell envelope proteins, including the phosphate permease systems, compared with WT. In vitro COFRADIC studies were performed to identify the specificity profiles of the recombinant proteases rHhoA, rHhoB or rHtrA added to the Synechocystis WT proteome. The combined in vivo and in vitro N-terminal COFRADIC datasets propose RbcS as a natural substrate for HhoA, PsbO for HhoB and HtrA and Pbp8 for HtrA. We therefore suggest that each Synechocystis Deg protease protects the cell through different, but connected mechanisms

  • 30. Thorbjornsen, T
    et al.
    Villand, P
    Kleczkowski, Leszek A
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Olsen, O A
    A single gene encodes two different transcripts for the ADP-glucose pyrophosphorylase small subunit from barley (Hordeum vulgare)1996In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 313, p. 149-154Article in journal (Refereed)
    Abstract [en]

    ADP-glucose pyrophosphorylase (AGPase), a heterotetrameric enzyme composed of two small and two large subunits, catalyses the first committed step of starch synthesis in plant tissues. In an attempt to learn more about the organization and expression of the small-subunit gene of AGPase, we have studied the small-subunit transcripts as well as the structure of the gene encoding these transcripts in barley (Hordeum vulgare L. cv. Bomi). Two different transcripts (bepsF1 and blps14) were identified: bepsF1 was abundantly expressed in the starchy endosperm but not in leaves, whereas blps14 was isolated from leaves but was also found to be present at a moderate level in the starchy endosperm. The sequences for the two transcripts are identical over approx. 90% of the length, with differences being confined solely to their 5' ends. In blps14, the unique 5' end is 259 nt long and encodes a putative plastid transit peptide sequence. For the 178-nt 5' end of bepsF1, on the other hand, no transit peptide sequence could be recognized. A lambda clone that hybridized to the AGPase transcripts was isolated from a barley genomic library and characterized. The restriction map has suggested a complex organization of the gene, with alternative exons encoding the different 5' ends of the two transcripts followed by nine exons coding for the common part of the transcripts. The sequence of a portion of the genomic clone, covering the alternative 5'-end exons as well as upstream regions, has verified that both transcripts are encoded by the gene. The results suggest that the small-subunit gene of barley AGPase transcribes two different mRNAs by a mechanism classified as alternative splicing.

  • 31.
    van Dijk, Jesper R.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Yamazaki, Yasuo
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Palmer, Ruth H.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Tumour-associated mutations of PA-TM-RING ubiquitin ligases RNF167/RNF13 identify the PA domain as a determinant for endosomal localization2014In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 459, no 1, p. 27-36Article in journal (Refereed)
    Abstract [en]

    Diverse cellular processes depend on endocytosis, intracellular vesicle trafficking, sorting and exocytosis, and processes that are regulated post-transcriptionally by modifications such as phosphorylation and ubiquitylation. The PA (protease-associated) domain E3 ligases, such as Godzilla(CG10277) in Drosophila melanogaster and RNF167 (RING finger protein 167) in humans, have been implicated in the regulation of cellular endosome trafficking. In the present study, we have characterized point mutations in the RING (really interesting new gene) domain of human RNF13 and RNF167, which have been identified in human tumour samples, that abrogate ubiquitin ligase activity as well as function. In the present study, we have also identified a functional role for the PA domain, which is required for endosomal localization of these proteins. Although the PA domain point mutations of RNF13 and RNF167 identified in human tumours are ligase active, the resultant mutant proteins are mislocalized within the cell. Thus the PA domain E3 ligases examined in the present study appear to require both E3 ligase activity as well as an intact PA domain to efficiently target and ubiquitylate their cellular substrates.

  • 32. Villand, P
    et al.
    Eriksson, Mats
    Samuelsson, Göran
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Carbon dioxide and light regulation of promoters controlling the expression of mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii1997In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 327, p. 51-57Article in journal (Refereed)
    Abstract [en]

    Nuclear genes coding for carbonic anhydrase, a major mitochondrial constituent in Chlamydomonas reinhardtii grown under limited CO2, were characterized. Two genes, ca1 and ca2, were found within 7 kb of genomic DNA, organized 'head to head' in a large inverted repeat. The DNA sequences for the two genes were very similar, even in the promoter regions and in introns, indicating that the repeat is a result of a recent duplication. To study gene regulation, elements from the upstream region of cal were fused to the arylsulphatase reporter gene. After transformation,the expression of arylsulphatase was regulated similarly to the endogenous ca1/ca2 genes, even when the promoter was trimmed down to 194 nt. Expression could not be detected when 5% CO2 was bubbled into the growth medium, but was induced within hours after transfer to air. The cal promoter was not induced in low light, but at intermediate light levels its activity was dependent on the irradiance. O-2 concentration had no effect on the promoter activity, indicating that photorespiratory metabolites are not triggering the response. The availability of cells transformed with a CO2-regulated reporter gene should facilitate further studies on the metabolic adaptations that occur in some green algae in response to the external CO2 level.

  • 33.
    Yasmin, Lubna
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Veesenmeyer, Jeffrey L
    Diaz, Maureen H
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ottmann, Christian
    Palmer, Ruth H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hauser, Alan R
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 427, no 2, p. 217-224Article in journal (Refereed)
    Abstract [en]

    14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells that play an important role in a multitude of signalling pathways. 14-3-3 proteins bind either to phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners [Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice and Mackintosh (2004) Biochem. J. 379, 395-408]. These interactions result in cell-cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. One example of a phosphorylation-independent interaction is the binding of 14-3-3 to ExoS (exoenzyme S), a bacterial ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. In the present study, we have utilized additional biochemical and infection analyses to define further the structural basis of the interaction between ExoS and 14-3-3. An ExoS leucine-substitution mutant dramatically reduced the interaction potential with 14-3-3 suggesting that Leu422, Leu423, Leu426 and Leu428 of ExoS are important for its interaction with 14-3-3, its enzymatic activity and cytotoxicity. However, ExoS substitution mutants of residues that interact with 14-3-3 through an electrostatic interaction, such as Ser416, His418, Asp424 and Asp427, showed no reduction in their interaction potential with 14-3-3. These ExoS substitution mutants were also as aggressive as wild-type ExoS at inducing cell death and to modify endogenous ExoS target within the cell. In conclusion, electrostatic interaction between ExoS and 14-3-3 via polar residues (Ser416, His418, Asp424 and Asp427) appears to be of secondary importance. Thus the interaction between the 'roof' of the groove of 14-3-3 and ExoS relies more on hydrophobic interaction forces, which probably contributes to induce cell death after ExoS infection and activation.

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