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  • 501. Vukojević, Vladana
    et al.
    Ming, Yu
    D'Addario, Claudio
    Hansen, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Schulz, Rüdiger
    Johansson, Björn
    Rigler, Rudolf
    Terenius, Lars
    Mu-opioid receptor activation in live cells2008Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 22, nr 10, s. 3537-3548Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Interaction of the mu-opioid receptor (MOP) with selected ligands was investigated in live cells using advanced imaging by confocal laser scanning microscopy integrated with fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. In PC12 cells stably transformed to express the fluorescently labeled MOP-enhanced green fluorescent protein construct, two pools of MOP were identified that could be discriminated by differences in their lateral mobility in the cell membrane. The majority of MOP receptors (80 +/- 10%) were characterized by a diffusion coefficient D-MOP,D-1 = (4 +/- 2) X 10(-11) m(2) s(-1), compared with the slowly moving fraction, D-MOP,D- 2 = (4 +/- 2) x 10(-12) m(2) s(-1). On stimulation with selected agonists ([D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin, enkephalin-heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe, morphine, and methadone), surface density of the MOP decreased, whereas the lateral mobility increased. In contrast, antagonists (naloxone and naltrexone) "froze" the receptor in the membrane, i.e., increased MOP surface density and decreased lateral mobility. Agonist activation was also accompanied by pronounced changes in the dynamics of plasma membrane lipids, as revealed by the general lipid marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate dye. The results provide new information about MOP activation in live cells at the molecular level, with a special focus on the dynamics of the intricate interplay between this receptor and the surrounding lipids.

  • 502.
    Wahlström, Karolina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Development and characterisation of protecting groups that enhance the solubility of synthetic peptides: Methods to improve the aqueous solubility of hydrophobic peptides2009Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Chemical synthesis of peptides is generally performed by solid phase peptide synthesis (SPPS). Although SPPS is a rapid and convenient method to prepare peptides, the major methodological problem in SPPS is related to solvation/solubility. Poor solvation of the peptide during the synthesis on solid phase will frequently lead to deletion peptides and if the peptide is poorly soluble after it has been cleaved from the resin, it can be difficult to purify by liquid chromatography. In order to increase the solubility of peptides, two new amino acid derivatives and a modification of the 2-hydroxy-4-methoxy-benzyl (Hmb) group, a protecting group for the peptide bond, have been developed. The Boc-N-methyl-N-[(2-methylamino)ethyl]carbamoyl (Boc-Nmec) group was used for protection of aromatic hydroxyl groups of tyrosine and the Hmb group. The Boc-4-(N-methyl-amino)butanoyl (Boc-Nmbu) group was used for protection of the indole nitrogen on tryptophan. The new derivatives were introduced into model peptides by standard protocol for Fmoc chemistry. The Boc protection of the Nmec/Nmbu group is removed during the cleavage of the peptide from the resin but the Nmec/Nmbu group is still attached to the peptide. The Nmec/Nmbu group contains a secondary amino group that is protonated at low pH and thereby increases the solubility during purification and handling of the peptide in aqueous solutions. By raising the pH the Nmec (pH 8)/Nmbu (pH 9) group is cleaved by an intramolecular cyclization reaction to give the fully deprotected peptide. Amyloid b (1-42) is a large peptide that is very difficult both to assemble on solid phase and to purify by HPLC.  It is shown that by using the Boc-Nmec protected dipeptides, amyloid b (1-42) could be synthesized by SPPS with only traces of by-products and that the peptide was easy to purify by HPLC. It is likely that the new protecting groups would be very useful in synthesis and handling of hydrophobic peptides.

     

     

  • 503.
    Wahlström, Karolina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Planstedt, Ove
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Undén, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    A carbamoyl-protective group for tyrosine that facilitatespurification of hydrophobic synthetic peptides2008Inngår i: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 49, nr 23, s. 3779-3781Artikkel i tidsskrift (Fagfellevurdert)
  • 504.
    Wahlström, Karolina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Planstedt, Ove
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Undén, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Synthesis and purification of aggregation-prone hydrophobicpeptides by the incorporation of an Fmoc dipeptide withthe peptide bond protected with a modified 2-hydroxy-4-methoxybenzyl (Hmb) group2008Inngår i: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 49, nr 24, s. 3921-3924Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The dipeptide Fmoc-Val-(2-hydroxy-4-methoxybenzyl)Gly-OBzl was synthesized and the 2-hydroxyl group carbamoylated to give a Boc-N(CH(3))CH(2)CH(2)N(CH(3))CO-, (Boc-Nmec-) modification of the 2-hydroxy-4-methoxybenzyl (Hmb) group. After catalytic hydrogenation and purification, the resulting dipeptide Fmoc-Val-(Boc-Nmec-Hmb)Gly-OH was used in solid phase peptide synthesis. During treatment with TFA, the peptide was released from the resin and the Boc group cleaved. The peptide could then be purified with an alkylated peptide bond carrying a cationic charge that both increased the solubility of the peptide during the purification steps and facilitated analysis by MALDI-TOF mass spectrometry. The Nmec group was cleaved by intramolecular cyclization under slightly alkaline conditions, followed by cleavage of the Hmb group by TFA to give the fully deprotected peptide.

  • 505.
    Wahlström, Karolina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Undén, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    A new protecting group for tryptophan in solid-phase peptide synthesis which protectsagainst acid-catalyzed side reactions and facilitates purification by HPLC2009Inngår i: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 50, nr 24, s. 2976-2978Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The indole nucleus of Z-Trp-OBzl is modified by acylation of the indole nitrogen using Boc-N-methyl butyric acid followed by catalytic hydrogenation and introduction of the Fmoc group. The resulting derivative, Fmoc-Trp(Boc-Nmbu)-OH, is incorporated into peptide chains via solid-phase peptide synthesis (SPPS). After assembly of the peptide chain, the Boc group is cleaved by treatment with TFA. The peptide is isolated with the tryptophan residue modified with a cationic 4-(N-methylamino) butanoyl group, which improves the solubility of the peptide during HPLC purification. On treatment of the purified peptide at pH 9.5, the Nmbu group undergoes an intramolecular cyclization reaction; this results in the fully deprotected peptide and N-methylpyrrolidone.

  • 506.
    Wahlström, Karolina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Undén, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Synthesis of Amyloid β (1-42) with protected peptide bondsManuskript (preprint) (Annet vitenskapelig)
  • 507.
    Wanderoy, Maria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Up-regulation of dopamine D₂ receptors: in vitro and in vivo studies1998Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In the central nervous system (CNS) dopaminergic and dopaminoceptive neurons have the ability to respond to variations in dopamine levels by for example adjusting their dopamine receptor levels. One of the most well known phenomena in this respect is that long-term blockade of D2 receptors with antipsychotic drugs leads to an increase in striatal D2 receptor density of experimental animals as well as of schizophrenic patients as measured in both post-mortem studies and in vivo positron emission tomography studies of brain. Whether such a response underlies schizophrenic symptoms, is an effect of drug treatment, or both has yet to be clarified.

    Knowledge of underlying molecular mechanisms of D2 receptor up-regulation is sparse due to the difficulty in examining such effects both in vivo and in vitro. This work mainly describes a way to overcome these obstacles by: (1) presenting a cellular system where increased D2 receptor levels and a supersensitive D2 coupled functional response can be evoked by persistent "indirect" blockade of the receptor (2) identifying some of the molecular aspects underlying this up-regulation and (3) validating parts of this cellular model in vivo in the rat striatum.

    By persistently stimulating the adenylyl cyclase enzyme with forskolin in mouse Ltk- fibroblast cells stably transfected with the human long D2 receptor (D2L) (and thereby mimicking long-term D2 receptor block at the level of intracellular cAMP), D2 receptors could be up-regulated by 43%. A supersensitive response to dopamine was found in parallel. The increase in receptor number was even larger (105%) for the short D2 receptor variant. The forskolin-induced up-regulation of D2L receptors was found to be dependent on: (1) increases in intracellular cAMP levels (2) de novo protein synthesis (3) activation of protein kinase A (PKA) and (4) at least partially on an intact Gi/o protein pool. Forskolin infusion into rat lateral ventricles produced D2 receptor increases of 19% and D1 receptor decreases of 27%. In vivo effects in the rats following the forskolin infusion, most likely a function of the D2 receptor up-regulation, were shown in both behavioral and biochemical experiments. The relevance of studying D2 receptor up-regulation was strengthened by findings where subchronic treatment of rats with the atypical antipsychotic remoxipride resulted in D2 receptor up-regulation in brain regions that have receptor levels that are 38-fold lower than in the striatum. Parallel control treatments with the typical antipsychotic haloperidol gave similar results.

    In conclusion, the work presents an in vitro possibility for studying molecular mechanisms of D2 receptor up-regulation. The cellular model has been characterized in several respects and partly validated in an in vivo system. There seems to be good reason to pursue these issues further as the new generation of atypical antipsychotics are also likely to target and affect D2 receptor numbers in the brain.

  • 508.
    Webling, Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Galanin receptor ligands: Design, synthesis, characterization and biological effects2016Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Galanin is a 29/30 amino acid long bioactive peptide discovered over 30 years ago when C-terminally amidated peptides were isolated from porcine intestines. The name galanin originates from a combination of the first and last amino acids - G from glycine and the rest from alanine. The first 15 amino acids are highly conserved among species which indicates that the N-terminus is important for receptor recognition and subsequent binding. Galanin exerts its effects by binding to three different G-protein coupled receptors, which all differ in regional distribution, the affinity for shortened galanin fragments, as well as the G-protein signaling cascade used. At the time of publication, galanin was found to cause muscle contraction as well as hyperglycemia.  Over the years, galanin has been reported to be involved in a wide variety of biological and pathological functions, for example epilepsy, food intake and depression.

    Determining the specific involvement of the three different galanin receptors in several biological and pathological processes is limited by the small amount of galanin receptor selective/specific ligands available as research tools. Furthermore, the fast degradation of peptides limits the administration routes in animal studies.

    This thesis aims at developing new galanin receptor-selective ligands to help delineate the involvement of the three different galanin receptors also known as the galaninergic system.

    Paper 1 demonstrates that the neuroprotective effects of galanin in a kainic acid induced excitotoxic animal model was mediated through galanin receptor 1. Furthermore, a new robust protocol for evaluating G-protein signaling using a label-free real time impedance technique was presented and compared to two different classical second-messenger assays.

    Paper 2 presents a series of systemically active galanin receptor 2 selective ligands subsequently evaluated in two different depression-like animal models.

    In conclusion, this thesis presents six new galanin ligands, which can be used to evaluate the galaninergic system as well as to investigate the possible use of peptides as pharmaceuticals.

  • 509.
    Webling, Kristin E
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Design, Synthesis and Characterization of Galanin Receptor Selective Ligands2017Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Galanin is a 29/30 amino acid long bioactive peptide discovered over 30 years ago when C-terminally amidated peptides were isolated from porcine intestines. The name galanin originates from a combination of the first and last amino acids - G from glycine and the rest from alanine. The first 15 amino acids are highly conserved throughout species, which indicates that the N-terminus is important for receptor recognition and binding. Galanin exerts its effects by binding to three different G protein-coupled receptors, which all differ according to regional distribution, the affinity for shortened galanin fragments, as well as the intracellular G-protein signaling cascade used. When first discovered, galanin was found to cause muscle contraction as well as hyperglycemia.  Over the years, galanin has been reported to be involved in a wide variety of biological functions, for example food intake and neurogenesis, and pathological functions, for example epilepsy and depression.

    Determining the specific involvement of the three different galanin receptors in biological and pathological processes is limited by the small amount of galanin receptor selective/specific ligands available as research tools. Furthermore, the fast degradation of peptides limits the administration routes in animal studies.

    This thesis aims at developing new galanin receptor-selective ligands to help delineate the involvement of the three different galanin receptors.

    Paper 1 presents the shortest galanin fragment with a galanin receptor 2 specific binding preference where only a single amino acid substitution was made, Ala5Ser in galanin (2-11). In addition, G-protein coupled receptor signaling were evaluated through both a classical second messenger assay and a real-time label-free technique in cells overexpressing the receptor as well as low receptor expression.

    Paper 2 demonstrates that the neuroprotective effects of galanin in a kainic acid-induced excitotoxic animal model were mediated through galanin receptor 1. Furthermore, a new robust protocol for evaluating G-protein signaling using a label-free real time impedance technique was presented and compared to two different classical second-messenger assays.

    Paper 3 presents a series of systemically active galanin receptor 2 selective ligands subsequently evaluated in two different depression-like animal models.

    Paper 4 investigates a mutated form of human galanin which was found in epilepsy patients and binding and signaling properties of the mutated associated ligand p.(A39E) was examined.

    In conclusion, this thesis presents the discovery of eight new galanin ligands, which can be used to evaluate the galaninergic system as well as to help investigate the possible use of peptides as pharmaceuticals in different diseases.

  • 510.
    Webling, Kristin E. B.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bartfai, Tamas
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Galanin receptors and ligands2012Inngår i: Frontiers in Endocrinology, ISSN 1664-2392, E-ISSN 1664-2392, Vol. 3, nr 146, s. 1-14Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The neuropeptide galanin was first discovered 30 years ago. Today, the galanin family consists of galanin, galanin-like peptide (GALP), galanin-message associated peptide (GMAP), and alarin and this family has been shown to be involved in a wide variety of biological and pathological functions. The effect is mediated through three GPCR subtypes, GalR1-3. The limited number of specific ligands to the galanin receptor subtypes has hindered the understanding of the individual effects of each receptor subtype. This review aims to summarize the current data of the importance of the galanin receptor subtypes and receptor subtype specific agonists and antagonists and their involvement in different biological and pathological functions.

  • 511.
    Webling, Kristin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Groves-Chapman, Jessica L.
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Saar, Indrek
    Lang, Andreas
    Sillard, Rannar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jakovenko, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kofler, Barbara
    Holmes, Philip V.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Pharmacological stimulation of GAL1R but not GAL2R attenuates kainic acid-induced neuronal cell death in the rat hippocampus2016Inngår i: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 58, s. 83-92Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The neuropeptide galanin is widely distributed in the central and peripheral nervous systems and part of a bigger family of bioactive peptides. Galanin exerts its biological activity through three G-protein coupled receptor subtypes, GAL1–3R. Throughout the last 20 years, data has accumulated that galanin can have a neuroprotective effect presumably mediated through the activation of GAL1R and GAL2R. In order to test the pharmaceutical potential of galanin receptor subtype selective ligands to inhibit excitotoxic cell death, the GAL1R selective ligand M617 and the GAL2R selective ligand M1145 were compared to the novel GAL1/2R ligand M1154, in their ability to reduce the excitotoxic effects of intracerebroventricular injected kainate acid in rats.

    The peptide ligands were evaluated in vitro for their binding preference in a competitive 125I-galanin receptor subtype binding assay, and G-protein signaling was evaluated using both classical signaling and a label-free real-time technique. Even though there was no significant difference in the time course or severity of the kainic acid induced epileptic behavior in vivo, administration of either M617 or M1154 before kainic acid administration significantly attenuated the neuronal cell death in the hippocampus. Our results indicate the potential therapeutic value of agonists selective for GAL1R in the prevention of neuronal cell death. 

  • 512.
    Webling, Kristin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lang, Andreas
    Saar, Indrek
    Kofler, Barbara
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Ala(5)-galanin (2-11) is a GAL2R specific galanin analogue2016Inngår i: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 60, s. 75-82Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    It is over 30years since the regulatory peptide galanin was discovered by Professor Mutt and co-workers. Galanin exerts its effects by binding to three galanin G-protein coupled receptors, namely GAL1R, GAL2R and GAL3R. Each galanin receptor has a different distribution in the central nervous system and the peripheral nervous system as well as distinctive signaling pathways, which implicates that the receptors are involved in different biological- and pathological effects. The delineation of the galaninergic system is however difficult due to a lack of stable, specific galanin receptor ligands. Herein, a new short GAL2R specific ligand, Ala(5)-galanin (2-11), is presented. The galanin (2-11) modified analogue Ala(5)-galanin (2-11) was tested in (125)I-galanin competitive binding studies for the three galanin receptors and the G-protein coupled receptor signaling properties was tested by the ability to influence second-messenger molecules like inositol phosphate and cyclic adenosine monophosphate. In addition, two different label-free real-time assays, namely EnSpire® based on an optical biosensor and xCELLigence® based on an electric biosensor, were used for evaluating the signaling properties using cell lines with different levels of receptor expression. Ala(5)-galanin (2-11) was subsequently found to be a full agonist for GAL2R with more than 375-fold preference for GAL2R compared to both GAL1R and GAL3R. The single amino acid substitution of serine to alanine at position 5 in the short ligand galanin (2-11) resulted in a ligand subsequently unable to bind neither GAL3R nor GAL1R, even at concentrations as high as 0.1mM.

  • 513.
    Wenehed, Viktoria
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Solyakov, Alexey
    Thylin, Ingrid
    Häggblom, Per
    Forsby, Anna
    Cytotoxic response of Aspergillus fumigatus-produced mycotoxins on growth medium, maize and commercial animal feed substrates2003Inngår i: Food and Chemical Toxicology, Vol. 41, s. 395-403Artikkel i tidsskrift (Fagfellevurdert)
  • 514. Wisén, Susanne
    et al.
    Sjögren, Tove
    Olin, Birgit
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Purification, crystallization and preliminary X-ray data of the transcription factor NtcA from the cyanobacterium Anabaena PCC 7120.2004Inngår i: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 60, nr Pt 5, s. 923-5Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    NtcA is a transcription factor that acts as a global nitrogen regulator in cyanobacteria. Cyanobacteria are photosynthetic prokaryotic organisms, some genera of which can fix nitrogen under conditions of nitrogen deprivation. NtcA from Anabaena PCC 7120 is a dimeric protein that consists of 223 amino acids with a molecular weight of 25 kDa per subunit. It belongs to the cAMP receptor-protein (CAP) family and is involved in the regulation of several of the genes acting in the nitrogen-fixation process. Here, the crystallization and preliminary X-ray data of NtcA are described. The crystallization was made possible by an improved purification method, which provides a stable NtcA protein at concentrations suitable for crystallization. The protein was crystallized using the hanging-drop method. Data were collected to 2.5 A resolution using synchrotron radiation and the crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 69.23, b = 69.23, c = 162.15 A, alpha = beta = gamma = 90 degrees. The phases necessary to solve the structure of NtcA could not be obtained by molecular replacement based on the CAP structure using various models.

  • 515. Yandek, Lindsay E.
    et al.
    Pokorny, Antje
    Florén, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Knoelke, Kristina
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Almeida, Paulo F. F.
    Mechanism of the cell-penetrating Peptide transportan 10 permeation of lipid bilayers2007Inngår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 92, nr 7, s. 2434-2444Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The mechanism of the interaction between the cell-penetrating peptide transportan 10 ( tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of peptide association with vesicles and peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence. The experimental kinetics were analyzed by directly fitting to the data the numerical solution of mathematical kinetic models. A very good global fit was obtained using a model in which tp10 binds to the membrane surface and perturbs it because of the mass imbalance thus created across the bilayer. The perturbed bilayer state allows peptide monomers to insert transiently into its hydrophobic core and cross the membrane, until the peptide mass imbalance is dissipated. In that transient state tp10 "catalyzes" dye efflux from the vesicle lumen. These conclusions are consistent with recent reports that used molecular dynamics simulations to study the interactions between peptide antimicrobials and phospholipid bilayers. A thermodynamic analysis of tp10 binding and insertion in the bilayer using water-membrane transfer hydrophobicity scales is entirely consistent with the model proposed. A small bilayer perturbation is both necessary and sufficient to achieve very good agreement with the model, indicating that the role of the lipids must be included to understand the mechanism of cell-penetrating and antimicrobial peptides.

  • 516.
    Yeste-Velasco, Marc
    et al.
    Facultat de Farmàcia, Universitat de Barcelona,.
    Alvira, Daniel
    Sureda, Francesc X
    Rimbau, Victor
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pallàs, Mercè
    Camins, Antoni
    Folch, Jaume
    DNA low-density array analysis of colchicine neurotoxicity in rat cerebellar granular neurons.2008Inngår i: Neurotoxicology, ISSN 0161-813X, E-ISSN 1872-9711, Vol. 29, nr 2, s. 309-17Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytoskeletal alteration is a key factor in neurodegenerative processes like Alzheimer's or Parkinson's disease. Colchicine is a microtubule-disrupting agent that binds to tubuline, inhibiting microtubule assembly, and which triggers apoptosis. The present research describes the transcriptional activation of molecules related to alternative forms of apoptosis, in an acute colchicine model of apoptosis in rat cerebellar granule neurons (CGNs). Treatment with colchicine up-regulated significantly the activity of genes related to oxidative stress: glutathione peroxidase 1 and catalase; altered significantly genes related to cell cycle control (cyclin D1 and cyclin-dependent kinase 2), genes related to classical apoptosis pathway (caspase 3) and a neuronal cell-related gene (pentraxin 1). Colchicine treatment also down-regulated the gene expression of calpain 1. In conclusion, our experiments demonstrate that the cell damage caused by exposure to colchicine activates the classical apoptosis pathway, but also promotes the up-regulation of several genes related to oxidative stress and cell cycle control. Present data may help to a better understanding of the molecular mechanisms involved in cytoskeletal degradation-induced apoptosis in neurons.

  • 517.
    Yu, Xin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    STUDIES OF FACTORS AFFECTING INTRACELLULAR TOXICITY OF THE SCA7 DISEASE PROTEIN ATAXIN - 7: FOCUS ON ATAXIN-7 DEGRADATION AND OXIDATIVE STRESS2011Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Spinocerebellar ataxia type 7 (SCA7) is one of nine neurodegenerative disorders caused by expansion of CAG/polyglutamine repeats. Proteins carrying expanded polyglutamine (polyQ) domains are suggested to be resistant to degradation and aggregate. Furthermore, a negative correlation between aggregation and toxicity has been shown. So far, little is known about the turn-over rate and degradation of the SCA7 disease protein ataxin-7 (ATXN7) and how this protein induces cellular toxicity. For the studies in this thesis work, we constructed stable inducible PC12 cell lines expressing GFP-tagged ATXN7 with 10 or 65 glutamines (Qs). Using these cell lines, we studied the turn-over of ATXN7 and the relationship between mutant ATXN7 and oxidative stress.

    We showed that ATXN7 with a normal glutamine repeat (ATXN7Q10-GFP) has a short half-life and is mainly degraded by the UPS. In cells expressing expanded ATXN7 (ATXN7Q65-GFP), aggregation and reduced viability was observed. The aggregation increased the half-life of mutant ATXN7. For expanded full-length ATXN7, UPS was still the main degradation pathway; however autophagy also played a role in clearance of soluble ATXN7 fragments and possibly in aggregated ATXN7 material. Moreover, activation of autophagy reduced the level of aggregation and ameliorated the toxicity in cells expressing mutant ATXN7. From this study, we could get the conclusion that although expansion of the polyQ repeat increases the stability of expanded ATXN7, the protein can still be degraded via both UPS and autophagy. Furthermore, stimulation of autophagy could ameliorate the expanded ATXN7 toxicity and could therefore be a potential therapeutic approach for SCA7.

    Regarding the role of oxidative stress we showed that expression of mutant ATXN7 leads to increased ROS levels and oxidative stress. Treatment with an antioxidant or blockage of NADPH oxidase complexes (NOX) decreased ATXN7 aggregation, the levels of ROS and ameliorated ATXN7 induced toxicity. Based on these results, we suggest that mutant ATXN7 cause increased ROS production from NOX and antioxidants treatment and or inhibition of NADPH-oxidase might potentially be used as a therapeutic strategy in SCA7.

  • 518.
    Yu, Xin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Studies of polyglutamine expanded Ataxin-7 toxicity2015Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant inherited neurodegenerative disease for which there is no cure. SCA7 belongs to the group of polyglutamine disorders, which are all caused by the expansion of a polyglutamine tract in different disease proteins. Common toxic mechanisms have been proposed for polyglutamine diseases; however the exact pathological mechanism(s) are still unclear.

    The aim of this thesis was to identify and characterize the molecular mechanisms by which polyglutamine expansion in the ATXN7 protein cause SCA7 and how this can be counteracted. We found that mutant ATXN7 can be degraded by the ubiquitin proteasome system (UPS) and autophagy, the two main cellular degradation pathways. However aggregation stabilized the protein against degradation. Moreover, we found that mutant ATXN7 blocked the induction of autophagy by interfering with p53 and the ULK1-ATG13-FIP200 complex. Pharmacological stimulation of autophagy ameliorated aggregation, as well as toxicity.

    We also found that oxidative stress plays an important role in mutant ATXN7 toxicity and that the oxidative stress is generated by activation of NADPH oxidase 1 (NOX1) complexes. Furthermore, we showed that the increased NOX1 activity, together with polyQ expanded ATXN7 mediated disruption of the transcription factor p53, results in metabolic alterations in SCA7 cells. The expression of key p53 regulated metabolic proteins like AIF, TIGAR and GLUT1 was altered in SCA7 cells and resulted in reduced mitochondrial respiration, a higher dependence on glycolysis and reduced ATP levels.

    In summary, our data indicate that mutant ATXN7 mediated dysregulation of p53, resulting in autophagic and metabolic alterations, could play a key role in SCA7 and possibly other polyglutamine diseases.

  • 519.
    Yu, Xin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ajayi, Abiodun
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Boga, Narasimha
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ström, Anna-Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Differential degradation of full-length and cleaved ataxin-7 fragments in a novel stable inducible SCA7 model2012Inngår i: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 47, nr 2, s. 219-233Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Spinocerebellar ataxia type 7 (SCA7) is one of nine neurodegenerative disorders caused by expanded polyglutamine repeats, and a common toxic gain-of-function mechanism has been proposed. Proteolytic cleavage of several polyglutamine proteins has been identified and suggested to modulate the polyglutamine toxicity. In this study, we show that full-length and cleaved fragments of the SCA7 disease protein ataxin-7 (ATXN7) are differentially degraded. We found that the ubiquitin-proteosome system (UPS) was essential for the degradation of full-length endogenous ATXN7 or transgenic full-length ATXN7 with a normal or expanded glutamine repeat in both HEK 293T and stable PC12 cells. However, a similar contribution by UPS and autophagy was found for the degradation of proteolytically cleaved ATXN7 fragments. Furthermore, in our novel stable inducible PC12 model, induction of mutant ATXN7 expression resulted in toxicity and this toxicity was worsened by inhibition of either UPS or autophagy. In contrast, pharmacological activation of autophagy could ameliorate the ATXN7-induced toxicity. Based on our findings, we propose that both UPS and autophagy are important for the reduction of mutant ataxin-7-induced toxicity, and enhancing ATXN7 clearance through autophagy could be used as a potential therapeutic strategy in SCA7.

  • 520.
    Yu, Xin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Muñoz-Alarcón, Andrés
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ajayi, Abiodun
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Webling, Kristin E.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Steinhof, Anne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ström, Anna-Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Inhibition of Autophagy via p53-Mediated Disruption of ULK1 in a SCA7 Polyglutamine Disease Model2013Inngår i: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 50, nr 3, s. 586-99Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Spinocerebellar ataxia type 7 (SCA7) is one of nine neurodegenerative disorders caused by expanded polyglutamine domains. These so-called polyglutamine (polyQ) diseases are all characterized by aggregation. Reducing the level of aggregating polyQ proteins via pharmacological activation of autophagy has been suggested as a therapeutic approach. However, recently, evidence implicating autophagic dysfunction in these disorders has also been reported. In this study, we show that the SCA7 polyglutamine protein ataxin-7 (ATXN7) reduces the autophagic activity via a previously unreported mechanism involving p53-mediated disruption of two key proteins involved in autophagy initiation. We show that in mutant ATXN7 cells, an increased p53-FIP200 interaction and co-aggregation of p53-FIP200 into ATXN7 aggregates result in decreased soluble FIP200 levels and subsequent destabilization of ULK1. Together, this leads to a decreased capacity for autophagy induction via the ULK1-FIP200-Atg13-Atg101 complex. We also show that treatment with a p53 inhibitor, or a blocker of ATXN7 aggregation, can restore the soluble levels of FIP200 and ULK1, as well as increase the autophagic activity and reduce ATXN7 toxicity. Understanding the mechanism behind polyQ-mediated inhibition of autophagy is of importance if therapeutic approaches based on autophagy stimulation should be developed for these disorders.

  • 521. Zaghloul, Eman M.
    et al.
    Gissberg, Olof
    Moreno, Pedro M. D.
    Siggens, Lee
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jørgensen, Anna S.
    Ekwall, Karl
    Zain, Rula
    Wengel, Jesper
    Lundin, Karin E.
    Smith, C. I. Edvard
    CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression2017Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, nr 9, s. 5153-5169Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation of HTT gene-associated RNA-polymerase II is demonstrated, suggestive of reduced transcription downstream the ON-targeted repeat. Different backbone chemistries were found to have a strong impact on the ON efficiency. We also successfully use different delivery vehicles as well as naked uptake of the ONs, demonstrating versatility and possibly providing insights for in vivo applications.

  • 522. Zeng, Fanyi
    et al.
    Peritz, Tiina
    Kannanayakal, Theresa J.
    Kilk, Kalle
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eiríksdóttir, Emelía
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eberwine, James
    A protocol for PAIR: PNA-assisted identification of RNA binding proteins in living cells2006Inngår i: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 1, nr 2, s. 920-927Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    All aspects of RNA metabolism are regulated by RNA-binding proteins (RBPs) that directly associate with the RNA. Some aspects of RNA biology such as RNA abundance can be readily assessed using standard hybridization technologies. However, identification of RBPs that specifically associate with selected RNAs has been more difficult, particularly when attempting to assess this in live cells. The peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology has recently been developed to overcome this issue. The PAIR technology uses a cell membrane–penetrating peptide (CPP) to efficiently deliver into the cell its linked PNA oligomer that complements the target mRNA sequence. The PNA will then anneal to its target mRNA in the living cell, and then covalently couple to the mRNA-RBP complexes subsequent to an ultraviolet (UV) cross-linking step. The resulting PNA-RNA-RBP complex can be isolated using sense oligonucleotide magnetic beads, and the RBPs can then be identified by mass spectrometry (MS). This procedure can usually be completed within 3 d. The use of the PAIR procedure promises to provide insight into the dynamics of RNA processing, transport, degradation and translation in live cells.

  • 523.
    Zetterström Fernaeus, Sandra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Changed iron metabolism and iron toxicity in scrapie-infected neuroblastoma cells2005Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Reactions and interactions of iron and oxygen can be both beneficial and detrimental to cells and tissues. Iron is mainly found in our blood where it functions as a mediator in the transport of oxygen to the cells and is further vital for the cellular respiration reducing the oxygen to water. The flexible redox state of iron makes it ideal to contribute in single electron transfers, but may also catalyze reactions with oxygen resulting in cell damaging reactive oxygen species (ROS). Normally the cells are protected against iron toxicity by controlling iron uptake and storage. When the intracellular demand for iron increases; the iron uptake is promoted by increasing the expression of transferrin receptor (TfR) and by decreasing the expression of the iron storage protein ferritin. Ferritin has a central role in the cellular iron detoxification by keeping it in a non reactive but still bioavailable form. However, in neurodegenerative diseases like in Alzheimer’s and Parkinson’s disease the iron storage capacity is disturbed and iron induced oxidative stress adds to the pathology of the diseases. The role of iron and its possible contribution to the pathology of prion diseases, like Creutzfeldt-Jakob disease, is less explored. In the first three studies of this thesis, the iron metabolism and the mutual relation between iron and oxygen are studied in scrapie-infected mouse neuroblastoma cells (ScN2a) as compared to control cells (N2a). In the fourth study we have analyzed the expression of ferritin and TfR in response to inflammation by treating the cells with the bacterial endotoxin lipopolysaccharide (LPS). LPS promotes the expression of inducible nitric oxide synthase (iNOS), a producer of nitric oxide (NO), a well known regulator of the iron metabolism.

    In the first study, the scrapie infection was found to reduce the iron levels, to reduce the mRNA and protein levels of ferritin and the TfR. In addition, reduced levels and activities of the iron regulatory proteins 1 and 2 were observed as compared to the uninfected N2a cells.

    In the second study, the addition of iron to the cell medium strongly increased the level of ROS and decreased the cell viability of the ScN2a cells, whereas the N2a cells were unaffected. The ferritin expression in N2a cells in response to the iron treatment was strongly increased and the concomitant measurement of the labile iron pool (LIP) revealed the LIP to be normalized within four hours. In the ScN2a cells the induction of ferritin expression was lower resulting in elevations in LIP that lasted up to 16 h, indicating that the increased ROS levels were iron catalyzed.

    In the third study, the cells were challenged with hydrogen peroxide (H2O2) to elevate the oxidative stress and to analyze the effects on the LIP and cell viability. The ScN2a cells were sensitive to the increased oxidative stress according to the cell viability test, and responded to the treatment with marked increase in the LIP levels, probably derived from an intra-cellular source. The cell viability could be reset by the co-addition of an iron chelator to the cell media. The N2a cells did not elevate the LIP and resisted higher concentrations of H2O2 than the ScN2a cells, according to the cell viability assay.

    In the fourth study, the LPS treatment resulted in increased mRNA levels of the heavy chain of ferritin, increased the protein levels of ferritin light chain and decreased the protein levels of the TfR in N2a cells, but no effects were observed in the ScN2a cells. Co-treatment with LPS and the iNOS inhibitor aminoguanidine did not affect the LPS induced decrease of TfR in N2a cells, whereas the free radical scavenger N-acetyl-L-cysteine reversed the effect of LPS on TfR expression, indicating that the changes were mediated by an oxidative rather than a nitric oxide mechanism in the N2a cells.

  • 524.
    Zetterström, Maria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Role of interleukin-1 in fever and inflammation1998Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The proinflammatory cytokines interleukin (IL) -1α, IL-1β and IL-6 are key mediators in the host's response to injury and infection. One of the systemic responses elicited by these proinflammatory cytokines, when injected peripherally or centrally, is fever. Thus, these proteins may act as endogenous pyrogens.

    We have shown that for a functional IL-1 mediated febrile response in vivo, both IL-1 receptor type I (IL-1RI) and IL-1 receptor accessory protein (IL-1RAcP) are necessary components, as knockout (KO) mice, lacking either IL-1RI or IL-1RAcP proteins, do not mount febrile responses to rrIL-1 (25-50 μg/kg, i.p.) injections, whereas they responded with fever when challenged by LPS (50 μg/kg, i.p.). These results suggest that IL-1 induces fever via signalling through a receptor complex, consisting of both IL-1RI and IL-1RAcP. We have also shown that IL-1β cannot induce NFκB translocation to the nucleus in primary astrocyte cultures derived from IL-1RAcP KO mice, suggesting a signalling role for this protein in mouse astrocytes.

    When the regulation of interleukin-1β converting enzyme (ICE, caspase-1) was studied in rat after peripheral LPS treatment (2 mg/kg, i.p.), elevations were seen, on both mRNA levels and enzyme activity in the pituitary, whereas in adrenal glands, an increase of mRNA levels did not coincide with upregulated enzyme activity. However, LPS was further shown to differentially regulate ICE isoforms, some of which are supposed to act as endogenous inhibitors of ICE activity. Thus, ICE activity seems to be tightly controlled on a transcriptional level, where regulation of ICE isoforms play an important role, as well as at the post transcriptional level.

    We have also shown that treatment with a neurotoxic fragment of β-amyloid, βA(25-35), induces a reactive phenotype of rat primary astrocyte cultures. The progression of this reactive phenotype was shown to coincide with elevated mRNA levels of IL-1α and IL-6. In addition, βA(25-35) treatment of primary astrocyte cultures derived from IL-1RI KO mice, induced a hypersensitive IL-1α response and a decreased IL-6 response. IL-1a and IL-6 are therefore proposed to be important molecules for development of reactive gliosis, and we also propose that signalling via IL-1RI is necessary for a full-scale induction of IL-6 mRNA.

  • 525. Zhang, Jie
    et al.
    Shibata, Aya
    Ito, Mika
    Shuto, Satoshi
    Ito, Yoshihiro
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Abe, Hiroshi
    Morgenstern, Ralf
    Synthesis and Characterization of a Series of Highly Fluorogenic Substrates for Glutathione Transferases, a General Strategy2011Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, nr 35, s. 14109-14119Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferases (GSTs) are used in biotechnology applications as fusion partners for facile purification and are also overexpressed in certain tumors. Consequently, there is a need for sensitive detection of the enzymes. Here we describe a general strategy for the synthesis and characterization of novel fluorogenic substrates for GSTs. The substrates were synthesized by introducing an electrophilic sulfonamide linkage to fluorescent molecules containing an amino group [e.g., 2,4-dinitrobenzenesulfonamide (DNs) derivatives of coumarin, cresyl violet, and rhodamine]. The derivatives were essentially nonfluorescent, and upon GST catalyzed cleavage of the dinitrobenzenesulfonamide, free fluorophore is released (and 1-glutathionyl-2,4-dinitrobenzene + SO(2)). All the coumarin-, cresyl violet- and rhodamine-based fluorogenic probes turned out to be good substrates for most GSTs, especially for GSTA(1-1), in terms of strong fluorescence increases (71-1200-fold), high k(cat)/K(m) values (10(4)-10(7) M(-1) s(-1)) and significant rate enhancements (10(6)-10(9)-fold). The substrates were successfully applied to quantitate very low levels of GST activity in cell extracts and DNs-cresyl violet was also successfully applied to the imaging of microsomal MGST(1) activity in living cells. The cresyl violet stained cells retained their fluorescence after fixation, which is a very useful property. In summary, we describe a general and versatile strategy to generate fluorogenic GST substrates, some of them providing the most sensitive assays so far described for GSTs.

  • 526. Zhang, Wei
    et al.
    Dourado, Daniel F. A. R.
    Fernandes, Pedro Alexandrino
    Ramos, Maria Joao
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Multidimensional epistasis and fitness landscapes in enzyme evolution2012Inngår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 445, s. 39-46Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The conventional analysis of enzyme evolution is to regard one single salient feature as a measure of fitness, expressed in a milieu exposing the possible selective advantage at a given time and location. Given that a single protein may serve more than one function, fitness should be assessed in several dimensions. In the present study we have explored individual mutational steps leading to a triple-point-mutated human GST (glutathione transferase) A2-2 displaying enhanced activity with azathioprine. A total of eight alternative substrates were used to monitor the diverse evolutionary trajectories. The epistatic effects of the imitations on catalytic activity were variable in sign and magnitude and depended on the substrate used, showing that epistasis is a multidimensional quality. Evidently, the multidimensional fitness landscape can lead to alternative trajectories resulting in enzymes optimized for features other than the selectable markers relevant at the origin of the evolutionary process. In this manner the evolutionary response is robust and can adapt to changing environmental conditions.

  • 527. Zhang, Wei
    et al.
    Dourado, Daniel F. A. R.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden; The Scripps Research Institute, USA.
    Evolution of the active site of human glutathione transferase A2-2 for enhanced activity with dietary isothiocyanates2015Inngår i: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1850, nr 4, s. 742-749Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Organic isothiocyanates (ITCs) are produced by plants, in which they are released from glucosinolates by myrosinase. ITCs are generally toxic and serve as a chemical defense against herbivorous insects and against infections by microorganisms. In mammalian tissues subtoxic concentrations of ITCs can provide protective effects against cancer and other diseases partially by induction of glutathione transferases (GSTs) and other detoxication enzymes. Thus, human consumption of edible plants rich in ITCs is presumed to provide health benefits. ITCs react with intracellular glutathione to form dithiocarbamates, catalyzed by GSTs. Formation of glutathione conjugates is central to the biotransformation of ITCs and leads to a route for their excretion. Clearly, the emergence of ITC conjugating activity in GSTs is essential from the biological and evolutionary perspective. Methods: In the present investigation an active-site-focused mutant library of GST A2-2 has been screened for enzyme variants with enhanced ITC activity. Results: Significantly superior activities were found in 34 of the approximately 2000 mutants analyzed, and the majority of the superior GSTs featured His and Gly residues in one of the three active-site positions subjected to mutagenesis. Conclusions: We explored the propensity of GSTs to obtain altered substrate selectivity and moreover, identified a specific pattern of mutagenesis in GST for enhanced PEITC detoxification, which may play an important role in the evolution of adaptive responses in organisms subjected to ITCs. General significance: The facile acquisition of enhanced ITC activity demonstrates that this important detoxication function can be promoted by numerous evolutionary trajectories in sequence space.

  • 528. Zhang, Wei
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    An improved dual-tube megaprimer approach for multi-site saturation mutagenesis2013Inngår i: World Journal of Microbiology & Biotechnology, ISSN 0959-3993, E-ISSN 1573-0972, Vol. 29, nr 4, s. 667-672Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Saturation mutagenesis is a powerful tool in protein engineering. Even though QuikChange site-directed mutagenesis method is dominantly used in laboratories, it could not be successfully applied to the generation of a focused mutant library of human glutathione transferase A2-2. In the present study, we further developed an improved versatile dual-tube approach of randomizing difficult-to-amplify targets, exhibiting significant improvement towards equal distribution of nucleotides at randomized sites compared to other published methods.

  • 529. Zhang, Wei
    et al.
    Modén, Olof
    Tars, Kaspars
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Structure-Based Redesign of GST A2-2 for Enhanced Catalytic Efficiency with Azathioprine2012Inngår i: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 19, nr 3, s. 414-421Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferase (GST) A2-2 is the most efficient human enzyme in the biotransformation of the prodrug azathioprine (Aza). The activation of Aza has therapeutic potential for possible use of GSTs in targeted enzyme-prodrug treatment of diseases. Based on the assumed catalytic mechanism and computational docking of Aza to the active site of the enzyme, active-site residues were selected for construction of focused mutant libraries, which were thereafter screened for Aza activity. Mutants with elevated Aza activity were identified, DNA sequenced, and the proteins purified. The two most active mutants showed up to 70-fold higher catalytic efficiency than the parental GST A2-2. The structure of the most active triple mutant (L107G/L108D/F222H) enzyme was determined by X-ray crystallography demonstrating significant changes in the topography of the active site facilitating productive binding of Aza as a substrate.

  • 530. Zhang, Zhenwen
    et al.
    Fang, Penghua
    He, Biao
    Guo, Lili
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Shi, Mingyi
    Zhu, Yan
    Bo, Ping
    Central Administration of Galanin Receptor 1 Agonist Boosted Insulin Sensitivity in Adipose Cells of Diabetic Rats2016Inngår i: Journal of Diabetes Research, ISSN 2314-6745, E-ISSN 2314-6753, artikkel-id 9095648Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Our previous studies testified the beneficial effect of central galanin on insulin sensitivity of type 2 diabetic rats. The aim of the study was further to investigate whether central M617, a galanin receptor 1 agonist, can benefit insulin sensitivity. The effects of intracerebroventricular administration of M617 on insulin sensitivity and insulin signaling were evaluated in adipose tissues of type 2 diabetic rats. The results showed that central injection of M617 significantly increased plasma adiponectin contents, glucose infusion rates in hyperinsulinemic-euglycemic clamp tests, GLUT4 mRNA expression levels, GLUT4 contents in plasma membranes, and total cell membranes of the adipose cells but reduced the plasma C-reactive protein concentration in nondiabetic and diabetic rats. The ratios of GLUT4 contents were higher in plasma membranes to total cell membranes in both nondiabetic and diabetic M617 groups than each control. In addition, the central administration of M617 enhanced the ratios of pAkt/Akt and pAS160/AS160, but not phosphorylative cAMP response element-binding protein (pCREB)/CREB in the adipose cells of nondiabetic and diabetic rats. These results suggest that excitation of central galanin receptor 1 facilitates insulin sensitivity via activation of the Akt/AS160 signaling pathway in the fat cells of type 2 diabetic rats.

  • 531. Zielinski, Jennifer
    et al.
    Kilk, Kalle
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Peritz, Tiina
    Kannanayakal, Theresa
    Miyashiro, Kevin Y.
    Eiríksdóttir, Emelía
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jochems, Jeanine
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eberwine, James
    In vivo identification of ribonucleoprotein–RNA interactions2006Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, nr 5, s. 1557-1562Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA–protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo. Specific regions of the 3ˈ and 5ˈ UTRs of ankylosis mRNA were targeted by antisense PNAs transported into cortical neurons by the cell-penetrating peptide transportan 10. An array of proteins was isolated in complex with or near the targeted regions of the ankylosis mRNA through UV-induced crosslinking of the annealed PNA–RNA–RBP complex. The first evidence for pharmacological modulation of these specific protein–RNA associations was observed. These data show that the PNA-assisted identification of the RBP technique is a reliable method to rapidly identify proteins interacting in vivo with the target RNA.

  • 532. Zirk, Katrin
    et al.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Vectorization of splice-correcting oligonucleotides with cell-penetrating peptides2013Inngår i: Chimica oggi, ISSN 0392-839X, E-ISSN 1973-8250, Vol. 31, nr 2, s. 12-15Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Personalized medicine approaches based on different gene therapy settings have gained much attention lately. In order to enforce successful gene therapy, genetic material needs to be delivered into cells. Nucleic acids and their analogues are unable to do so and thus require assistance to reach their site of action residing in the cytoplasm or nucleus. Here we give a short review on recent advancements in cell-penetrating peptide mediated delivery of splice-correcting oligonucleotides. We report on different cell-penetrating peptides applied for vectorization of splice-correcting oligonucleotides using both covalent conjugation and non-covalent nanoparticle formation approach. While covalent conjugation has gained extensive interest, there have also been great advances in non-covalent complex formation.

  • 533.
    Öjemalm, Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Higuchi, Takashi
    Jiang, Yang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    White, Stephen H.
    Suga, Hiroaki
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum2011Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, nr 31, s. E359-E364Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) Nature427:36–44; Zimmer J, et al. (2008) Nature455:936–943; Egea PF, Stroud RM (2010)Proc Natl Acad Sci USA 107:17182–17187]. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid’s contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal∕ðmol · Å2Þ, the solvation parameter for transfer from translocon to bilayer is 6 –10 cal∕ðmol · Å2Þ, pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a termodynamic partitioning model and insights into the physical properties of the translocon.

  • 534.
    Östlund, Pernilla
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindegren, Heléne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pettersson, Christina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bedecs, Katarina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Altered insulin receptor processing and function in scrapie-infected neuroblastoma cell lines2001Inngår i: Brain Research. Molecular Brain Research, ISSN 0169-328X, E-ISSN 1872-6941, Vol. 97, nr 2, s. 161-170Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The underlying neurochemical changes contributing to prion-induced neurodegeneration remain largely unknown. This study showsthat scrapie infection induced a 2-fold increase of insulin receptor (IR) protein and aberrantly processed IR b-chain in scrapie-infectedN2a neuroblastoma cells (ScN2a) as measured byWestern blot of immunoprecipitated IR, in the absence of increased IR mRNA. ElevatedIR protein level was further confirmed in an independently scrapie-infected neuroblastoma cell line N1E-115 (ScN1E-115). Proliferationstudies showed that the increased IR level in ScN2a did not result in an increased insulin-mediated cell growth compared to normal N2a125 cells. Binding studies indicated that this apparent paradox was due to a 65% decrease in specific [ I]insulin binding sites in ScN2a whencompared to the amount of immunoreactive IR, although the IR binding affinity was unchanged. Analysis of insulin stimulated IR tyrosinephosphorylation showed a slight but not significant reduction in ScN2a, when related to the increased level of immunoreactive IR.However, comparing the IR tyrosine phosphorylation to the loss of binding sites in ScN2a, we demonstrated an increased IR tyrosinephosphorylation of the remaining functional IR. In addition to these differences in IR properties, the basal extracellular signal regulatedkinase-2 (ERK2) phosphorylation detected by Western blot, was significantly elevated and the insulin stimulated ERK2 phosphorylationwas subsequently decreased in ScN2a. Together, these data show that scrapie infection affects the level and processing of the IR andsignal transduction mediated by the IR in neuroblastoma cells, as well as induces an elevated basal ERK2 phosphorylation. Aberrantregulation of neuroprotective receptors may contribute to neurodegeneration in prion diseases.

  • 535.
    Östlund, Pernilla
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindegren, Heléne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pettersson, Christina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bedecs, Katarina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie-infected neuroblastoma cells2001Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, nr 39, s. 36110-36115Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A growing body of evidence suggests that an altered level or function of the neurotrophic insulin-like growth factor-1 receptor (IGF-1R), which supports neuronal survival, may underlie neurodegeneration. This study has focused on the expression and function of the IGF-1R in scrapie-infected neuroblastoma cell lines. Our results show that scrapie infection induces a 4-fold increase in the level of IGF-1R in two independently scrapie-infected neuroblastomas, ScN2a and ScN1E-115 cells, and that the increased IGF-1R level was accompanied by increased IGF-1R mRNA levels. In contrast to the elevated IGF-IR expression in ScN2a, receptor binding studies revealed an 80% decrease in specific I-125-IGF-1-binding sites compared with N2a cells. This decrease in IGF-1R-binding sites was shown to be caused by a 7-fold decrease in IGF-1R affinity. Furthermore, ScN2a showed no significant difference in IGF-1 induced proliferative response, despite the noticeable elevated IGF-1R expression, putatively explained by the reduced IGF-1R binding affinity. Additionally, IGF-1 stimulated IGF-1R beta tyrosine phosphorylation showed no major change in the dose-response between the cell types, possibly due to altered tyrosine kinase signaling in scrapie-infected neuroblastoma cells. Altogether these data indicate that scrapie infection affects the expression, binding affinity, and signal transduction mediated by the IGF-1R in neuroblastoma cells. Altered IGF-1R expression and function may weaken the trophic support in scrapie-infected neurons and thereby contribute to neurodegeneration in prion diseases

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