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  • 1051.
    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 hippocampus2016Ingår i: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 58, s. 83-92Artikel i tidskrift (Refereegranskat)
    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. 

  • 1052.
    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 analogue2016Ingår i: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 60, s. 75-82Artikel i tidskrift (Refereegranskat)
    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.

  • 1053. Weibrecht, Irene
    et al.
    Lundin, Elin
    Kiflemariam, Sara
    Mignardi, Marco
    Grundberg, Ida
    Larsson, Chatarina
    Koos, Björn
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala universitet.
    Söderberg, Ola
    In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay2013Ingår i: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 8, nr 2, s. 355-372Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Analysis at the single-cell level is essential for the understanding of cellular responses in heterogeneous cell populations, but it has been difficult to perform because of the strict requirements put on detection methods with regard to selectivity and sensitivity (i.e., owing to the cross-reactivity of probes and limited signal amplification). Here we describe a 1.5-d protocol for enumerating and genotyping mRNA molecules in situ while simultaneously obtaining information on protein interactions or post-translational modifications; this is achieved by combining padlock probes with in situ proximity ligation assays (in situ PLA). In addition, we provide an example of how to design padlock probes and how to optimize staining conditions for fixed cells and tissue sections. Both padlock probes and in situ PLA provide the ability to directly visualize single molecules by standard microscopy in fixed cells or tissue sections, and these methods may thus be valuable for both research and diagnostic purposes.

  • 1054.
    Weidner, Jessica M.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Barragan, Antonio
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Tightly regulated migratory subversion of immune cells promotes the dissemination of Toxoplasma gondii2014Ingår i: International Journal of Parasitology, ISSN 0020-7519, E-ISSN 1879-0135, Vol. 44, nr 2, s. 85-90Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    While the spread of Toxoplasma gondii within the infected human or animal host is associated with pathology, the pathways of dissemination have remained enigmatic. From the time point of entry into the gut, to the quiescent chronic infection in the central nervous system, Toxoplasma is detected and surveyed by immune cells that populate the tissues, for example dendritic cells. Paradoxically, this protective migratory function of leukocytes appears to be targeted by Toxoplasma to mediate its dissemination in the organism. Recent findings show that tightly regulated events take place shortly after host cell invasion that promote the migratory activation of infected dendritic cells. Here, we review the emerging knowledge on how this obligate intracellular protozoan orchestrates the subversion of leukocytes to achieve systemic dissemination and reach peripheral organs where pathology manifests.

  • 1055.
    Westberg, Emelie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Hedebrant, Ulla
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Haglund, Johanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Alsberg, Tomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM).
    Eriksson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Seidel, Albrecht
    Törnqvist, Margareta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Conditions for sample preparation and quantitative HPLC/MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models2014Ingår i: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, nr 5, s. 1519-1530Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Stable adducts to serum albumin (SA) from electrophilic and genotoxic compounds/metabolites can be used as biomarkers for quantification of the corresponding in vivo dose. In the present study, conditions for specific analysis of stable adducts to SA formed from carcinogenic polycyclic aromatic hydrocarbons (PAH) were evaluated in order to achieve a sensitive and reproducible quantitative method. Bulky adducts from diolepoxides (DE) of PAH, primarily DE of benzo[a]pyrene (BPDE) and also DE of dibenzo[a,l]pyrene (DBPDE) and dibenzo[a,h]anthracene (DBADE), were used as model compounds. The alkylated peptides obtained after enzymatic hydrolysis of human SA modified with the different PAHDE were principally PAHDE-His-Pro, PAHDE-His-Pro-Tyr and PAHDE-Lys. Alkaline hydrolysis under optimised conditions gave the BPDE-His as the single analyte of alkylated His, but also indicated degradation of this adduct. It was not possible to obtain the BPDE-His as one analyte from BPDE-alkylated SA through modifications of the enzymatic hydrolysis. The BPDE-His adduct was shown to be stable during the weak acidic conditions used in the isolation of SA. Enrichment by HPLC or SPE, but not butanol extraction, gave good recovery, using Protein LoBind tubes. A simple internal standard (IS) approach using SA modified with other PAHDE as IS was shown to be applicable. A robust analytical procedure based on digestion with pronase, enrichment by HPLC or SPE, and analysis with HPLC/MS-MS electrospray ionisation was achieved. A good reproducibility (coefficient of variation (CV) 11 %) was obtained, and the achieved limit of detection for the studied PAHDE, using standard instrumentation, was approximately 1 fmol adduct/mg SA analysing extract from 5 mg SA.

  • 1056.
    Westerlund, Kristina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Moran, Sean D.
    Privett, Heidi K.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hay, Sam
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, Juri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gibney, Brian R.
    Tommos, Cecilia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Making a single-chain four-helix bundle for redox chemistry studies2008Ingår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, nr 11, s. 645-652Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The construction and characteristics of the stable and well-structured alpha W-4 protein are described. The 117-residue, single-chain protein has a molecular weight of 13.1 kDa and is designed to fold into a four-helix bundle. Experimental characterization of the expressed and purified protein shows a 69.8 +/- 0.8% helical content over a 5.5-10.0 pH range. The protein is thermostable with a T-M > 355 K and has a free energy of unfolding as measured by chemical denaturation of -4.7 kcal mol(-1) at 25 degrees C and neutral pH. One-dimensional (1D) proton and 2D N-15-HSQC spectra show narrow, well-dispersed spectral lines consistent with a uniquely structured alpha-helical protein. Analytical ultracentrifugation and NMR data show that the protein is monomeric over a broad protein concentration range. The 324 nm emission maximum of the unique Trp-106 is consistent with a sequestered position of the aromatic residue. Additionally, differential pulse voltammetry characterization indicates an elevated peak potential for Trp-106 when the protein is folded (pH range 7.0-8.5) relative to partly unfolded (pH range 11.4-13.2). The oxidation of Trp-106 is coupled to proton release as shown by a 53 +/- 3 mV/pH unit dependence of the peak potential over the 7.0-8.5 pH range.

  • 1057.
    Westman, Jacob
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Synthesis of oligosaccharides related to heparin and heparan sulphate and their binding to fibroblast growth factors1995Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 1058. Whitelam, Stephen
    et al.
    Geissler, Phillip L.
    Pronk, Sander
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Microscopic implications of S-DNA2010Ingår i: Physical review. E, ISSN 1539-3755, Vol. 82, nr 2, s. 21907-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recent experiments [J. van Mameren et al., Proc. Natl. Acad. Sci. U.S.A. 106, 18231 (2009)] provide a detailed spatial picture of overstretched DNA, showing that under certain conditions the two strands of the double helix separate at about 65 pN. It was proposed that this observation rules out the existence of an elongated, hybridized form of DNA (S-DNA). Here, we argue that the S-DNA picture is consistent with the observation of unpeeling during overstretching. We demonstrate that assuming the existence of S-DNA does not imply DNA overstretching to consist of the complete or near-complete conversion of the molecule from B to S form. Instead, this assumption implies in general a more complex dynamic coexistence of hybridized and unhybridized forms of DNA. We argue that such coexistence can rationalize several recent experimental observations.

  • 1059. Whittle, Andrew J.
    et al.
    Carobbio, Stefania
    Martins, Luis
    Slawik, Marc
    Hondares, Elayne
    Jesus Vazquez, Maria
    Morgan, Donald
    Csikasz, Robert I.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för fysiologi.
    Gallego, Rosalia
    Rodriguez-Cuenca, Sergio
    Dale, Martin
    Virtue, Samuel
    Villarroya, Francesc
    Cannon, Barbara
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för fysiologi.
    Rahmouni, Kamal
    Lopez, Miguel
    Vidal-Puig, Antonio
    BMP8B Increases Brown Adipose Tissue Thermogenesis through Both Central and Peripheral Actions2012Ingår i: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 149, nr 4, s. 871-885Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Thermogenesis in brown adipose tissue (BAT) is fundamental to energy balance and is also relevant for humans. Bone morphogenetic proteins (BMPs) regulate adipogenesis, and, here, we describe a role for BMP8B in the direct regulation of thermogenesis. BMP8B is induced by nutritional and thermogenic factors in mature BAT, increasing the response to noradrenaline through enhanced p38MAPK/CREB signaling and increased lipase activity. Bmp8b(-/-) mice exhibit impaired thermogenesis and reduced metabolic rate, causing weight gain despite hypophagia. BMP8B is also expressed in the hypothalamus, and Bmp8b(-/-) mice display altered neuropeptide levels and reduced phosphorylation of AMP-activated protein kinase (AMPK), indicating an anorexigenic state. Central BMP8B treatment increased sympathetic activation of BAT, dependent on the status of AMPK in key hypothalamic nuclei. Our results indicate that BMP8B is a thermogenic protein that regulates energy balance in partnership with hypothalamic AMPK. BMP8B may offer a mechanism to specifically increase energy dissipation by BAT.

  • 1060. Wickstrom, David
    et al.
    Wagner, Samuel
    Baars, Louise
    Ytterberg, A. Jimmy
    Klepsch, Mirjam
    van Wijk, Klaas J.
    Luirink, Joen
    de Gier, Jan-Willem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Consequences of Depletion of the Signal Recognition Particle in Escherichia coli2011Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 6, s. 4598-4609Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Thus far, the role of the Escherichia coli signal recognition particle (SRP) has only been studied using targeted approaches. It has been shown for a handful of cytoplasmic membrane proteins that their insertion into the cytoplasmic membrane is at least partially SRP-dependent. Furthermore, it has been proposed that the SRP plays a role in preventing toxic accumulation of mistargeted cytoplasmic membrane proteins in the cytoplasm. To complement the targeted studies on SRP, we have studied the consequences of the depletion of the SRP component Fifty-four homologue (Ffh) in E. coli using a global approach. The steady-state proteomes and the proteome dynamics were evaluated using one- and two-dimensional gel analysis, followed by mass spectrometry-based protein identification and immunoblotting. Our analysis showed that depletion of Ffh led to the following: (i) impaired kinetics of the biogenesis of the cytoplasmic membrane proteome; (ii) lowered steady-state levels of the respiratory complexes NADH dehydrogenase, succinate dehydrogenase, and cytochrome bo(3) oxidase and lowered oxygen consumption rates; (iii) increased levels of the chaperones DnaK and GroEL at the cytoplasmic membrane; (iv) a sigma(32) stress response and protein aggregation in the cytoplasm; and (v) impaired protein synthesis. Our study shows that in E. coli SRP-mediated protein targeting is directly linked to maintaining protein homeostasis and the general fitness of the cell.

  • 1061.
    Wieslander, Lars
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Björk, Petra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Nucleocytoplasmic mRNP export is an integral part of mRNP biogenesis2011Ingår i: Chromosoma, ISSN 0009-5915, E-ISSN 1432-0886, Vol. 120, nr 1, s. 23-38Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Nucleocytoplasmic export and biogenesis of mRNPs are closely coupled. At the gene, concomitant with synthesis of the pre-mRNA, the transcription machinery, hnRNP proteins, processing, quality control and export machineries cooperate to release processed and export competent mRNPs. After diffusion through the interchromatin space, the mRNPs are translocated through the nuclear pore complex and released into the cytoplasm. At the nuclear pore complex, defined compositional and conformational changes are triggered, but specific cotranscriptionally added components are retained in the mRNP and subsequently influence the cytoplasmic fate of the mRNP. Processes taking place at the gene locus and at the nuclear pore complex are crucial for integrating export as an essential part of gene expression. Spatial, temporal and structural aspects of these events have been highlighted in analyses of the Balbiani ring genes

  • 1062.
    Wigerius, Michael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Roles of mammalian Scribble in polarity signaling, virus offense and cell-fate determination2010Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Mammalian Scribble is a target for proteins encoded by human papilloma virus, retro- and flaviviruses. Tick-borne encephalitis virus (TBEV) is a flavivirus that have evolved distinct strategies to escape antiviral responses. Information of how flaviviruses intrude on cell integrity comes from understanding of the roles that host-factors play when they interfere with viruses. The first part of this thesis describes a novel interaction between the TBEVNS5 protein and Scribble. The importance of the interaction was demonstrated by RNAi-mediated depletion of Scribble, which prevented suppression of JAK-STAT signaling by NS5. Together, these results define Scribble as a novel target for NS5.

    TBEV is known to cause central nervous system disease TBE in humans that can lead to cognitive dysfunction. A unifying theme in CNS related diseases are defects in neuronal extensions. We therefore addressed the effects of TBEV expression in PC12 cell differentiation, which is characterized by extensive neurite growth. Our data show that TBEVNS5 suppresses neurite outgrowth through the Rho GTPase Rac1. These findings provide evidence that Rac1 is an indirect target of NS5 in neurite inhibition. Scribble was recently implicated in spine morphogenesis. Thus, we tested the role of Scribble in neurite elongation. Depletion of Scribble in PC12 cells, reduced neurite density but increased length of those remaining. Moreover, Scribble bound components in the Ras/ERK cascade in a growth factor dependent manner. Together, these results demonstrate that Scribble controls neurite elongation by scaffolding MAPK components. Moreover, as loss of dendritic spines, actin-rich protrusions on neurons, is a feature in cognitive dysfunction we speculate that cognitive dysfunction in TBE might involve disturbed Scribble expression by NS5.

    We also investigated the binding between NS1 of Influenza A virus and Scribble. The PDZ domains of Scribble are usually selective for specific C-terminal motifs in proteins. Because NS1 has a canonical PDZ motif we tested if binding to Scribble depends on this motif. We found that Scribble binds NS1; the association is dependent on the NS1 C-terminus that is recognized by PDZ3-4 of Scribble. Together, these results suggest that Scribble is a target for the H5N1 NS1 protein 

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  • 1063. Wiklund, Magda-Lena
    et al.
    Steinert, Stefanie
    Junell, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Hultmark, Dan
    Stoven, Svenja
    The N-terminal half of the Drosophila Rel/NF-kappa B factor Relish, REL-68, constitutively activates transcription of specific Relish target genes2009Ingår i: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 33, nr 5, s. 690-696Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Rel/NF-kappa B transcription factor Relish is a major regulator of the antimicrobial response in Drosophila. Upon immune challenge, Relish is cleaved to generate two fragments, the DNA-binding transcription factor REL-68 and the I kappa B-like REL-49. Using transgenic fly strains we show here that overexpression of REL-68 separately from REL-49 is sufficient to activate strong constitutive transcription of the Diptericin gene, but little constitutive or inducible transcription of Attacin and Cecropin, two other Relish target genes. Their transcription may therefore require additional modifications of Relish. However, phosphorylation of the conserved serine residue S431 is not involved in such modifications. This is unlike p65 and Dorsal, which are modulated by phosphorylation at their homologous site. In contrast to other I kappa B proteins, overexpression of REL-49 had no inhibitory effect on Relish-dependent transcription. Instead, we propose that the C-terminal I kappa B-like domain executes a scaffolding and recruiting function for full activation of Relish.

  • 1064.
    Wikström, Malin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Synthesis and protein curing abilities of membrane glycolipids2006Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    There are many types of membrane lipids throughout Nature. Still little is known about synthesizing pathways and how different lipids affect the embedded membrane proteins. The most common lipids are glycolipids since they dominate plant green tissue. Glycolipids also exist in mammal cells as well as in most Gram-positive bacteria. Glycosyltransferases (GTs) catalyze the final enzymatic steps for these glycolipids. In the bacteria Acholeplasma laidlawii and Streptococcus pneumonie and in the plant Arabidopsis thaliana, GTs for mono-/di-glycosyl-diacylglycerol (-DAG) are suggested to be regulated to keep a certain membrane curvature close to a bilayer/nonbilayer phase transition. The monoglycosylDAGs are nonbilayer-prone with small headgroups, hence by themselves they will not form bilayer structures.

    Here we have determined the genes encoding the main glycolipids of A. laidlawii and S. pneumonie. We have also shown that these GTs belong to a large enzyme group widely spread in Nature, and that all four enzymes are differently regulated by membrane lipids. The importance of different lipid properties were traced in a lipid mutant of Escherichia coli lacking the major (75 %), nonbilayer-prone/zwitterionic, lipid phosphatidylethanolamine. Introducing the genes for the GTs of A. laidlawii and two analogous genes from A. thaliana yielded new strains containing 50 percent of glyco-DAG lipids. The monoglyco-DAG strains contain significant amounts of nonbilayer-prone lipids while the diglyco-DAG strains contain no such lipids. Comparing these new strains for viability and the state of membrane-associated functions made it possible to connect different functions to certain lipid properties. In summary, a low surface charge density of anionic lipids is important in E.coli membranes, but this fails to be supportive if the diluting species have a too large headgroup. This indicates that a certain magnitude of the curvature stress is crucial for the membrane bilayer in vivo.

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  • 1065.
    Wikström, Malin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Xie, J.
    Bogdanov, M.
    Mileyovskaya, E.
    Heacock, P.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dowhan, W.
    Monoglucosyldiacylglycerol, a foreign lipid, can substitute for phosphatidylethanolamine in essential membrane-associated functions in Escherichia coli2004Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 11, s. 10484-10493Artikel i tidskrift (Refereegranskat)
  • 1066.
    Wilhelmsson, Christine
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Proteomics of the Drosophila hemolymph clot and the function of transglutaminase2009Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Insects rely on a fast and effective coagulation and wound response to avoid loss of body fluids and immobilize pathogens. Arthropod coagulation is in some respect equivalent to vertebrate coagulation but most factors and the regulation of coagulation systems seem not to be phylogenetically conserved. To get a more complete picture of insect clotting we studied the molecular and functional nature of Drosophila hemolymph coagulation.

    We developed new proteomic methods to collect Drosophila clotting factors. Several candidate factors were identified, including both predicted and novel clot proteins. Five putative TG (transglutaminase) substrates were found and we could also demonstrate that the clot is involved in immobilization of bacteria. Further investigating the role of TG we found TG to be important for Drosophila coagulation and that Fondue is a major substrate of the enzyme. Using fon RNAi knockdown we showed that Fondue affects the physical properties of the clot. A fon-GFP fusion construct was generated to follow its expression. The cuticle and the clot were labelled suggesting that Fondue is incorporated into both cuticle and clot. Clot properties and composition were affected by inhibiting TG chemically (MDC) and genetically (RNAi). Moreover, interaction between Fondue and Eig71Ee was demonstrated. Previous results indicated that coagulation could have an immune function. In hemolymph preparations, containing selected microorganisms, small deposits were seen on the microbial surfaces. The contents of these were investigated, revealing the presence of procoagulants. The targeting of microbes is instant and depends on TG and its substrates. Entomopathogenic nematode infections were performed to validate the functional importance of TG. TG RNAi knockdown larvae showed increased mortality, supporting an immune function for TG. Altogether, our data provide a more comprehensive picture of Drosophila immunity, and may further improve the understanding of innate immunity in general.

  • 1067.
    Wincent, Emma
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    6-Formylindolo[3,2-b]carbazole (FICZ) metabolism2007Konferensbidrag (Övrig (populärvetenskap, debatt, mm))
  • 1068.
    Wincent, Emma
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Bengtsson, Johanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Bardbori, Afshin Mohammadi
    Alsberg, Tomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM).
    Luecke, Sandra
    Rannug, Ulf
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Rannug, Agneta
    Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor2012Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, nr 12, s. 4479-4484Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Altered systemic levels of 6-formylindolo[3,2-b]carbazole (FICZ), an enigmatic endogenous ligand for the aryl hydrocarbon receptor (AHR), may explain adverse physiological responses evoked by small natural and anthropogenic molecules as well as by oxidative stress and light. We demonstrate here that several different chemical compounds can inhibit the metabolism of FICZ, thereby disrupting the autoregulatory feedback control of cytochrome P4501 systems and other proteins whose expression is regulated by AHR. FICZ is both the most tightly bound endogenous agonist for the AHR and an ideal substrate for cytochrome CYP1A1/1A2 and 1B1, thereby also participating in an autoregulatory loop that keeps its own steady-state concentration low. At very low concentrations FICZ influences circadian rhythms, responses to UV light, homeostasis associated with pro-and anti-inflammatory processes, and genomic stability. Here, we demonstrate that, if its metabolic clearance is compromised, femtomolar background levels of this compound in cell-culture medium are sufficient to up-regulate CYP1A1 mRNA and enzyme activity. The oxidants UVB irradiation and hydrogen peroxide and the model AHR antagonist 3'-methoxy-4'-nitroflavone all inhibited induction of CYP1A1 enzyme activity by FICZ or 2,3,7,8-tetrachlorodibenzo-p-dioxin, thereby subsequently elevating intracellular levels of FICZ and activating AHR. Taken together, these findings support an indirect mechanism of AHR activation, indicating that AHR activation by molecules with low affinity actually may reflect inhibition of FICZ metabolism and raising questions about the reported promiscuity of the AHR. Accordingly, we propose that prolonged induction of AHR activity through inhibition of CYP1 disturbs feedback regulation of FICZ levels, with potential detrimental consequences.

  • 1069. Witkowska, Ewa
    et al.
    Nowakowski, Michal
    Oleszczuk, Marta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Filip, Katarzyna
    Ciszewska, Malgorzata
    Chung, Nga N.
    Schiller, Peter W.
    Wojcik, Jacek
    Izdebski, Jan
    Ureido group containing cyclic dermorphin(1-7) analogues: synthesis, biology and conformation2007Ingår i: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 13, nr 8, s. 519-528Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Six cyclic peptides related to dermorphin(1-7) have been synthesized. The synthesis of linear peptides containing diamino acid residues in positions 2 and 4 was carried out on a 4-methylbenzhydrylamine resin, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid agonist activities were observed, depending on the size of the ring. The results were compared with those obtained earlier for 1-4 dermorphin analogues. The conformations of all six dermorphin analogues were studied. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. On the basis of NMR data, an ensemble of conformations was obtained for each peptide. The opioid activity profiles of the compounds are discussed in the light of the structural data.

  • 1070. Wojcik, Anna
    et al.
    Broclawik, Ewa
    Siegbahn, Per E. M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum.
    Borowski, Tomasz
    Mechanism of Benzylic Hydroxylation by 4-Hydroxymandelate Synthase: A Computational Study2012Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, nr 47, s. 9570-9580Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hydroxymandelate synthase (HMS) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) are highly related enzymes using the same substrates but catalyzing hydroxylation reactions yielding different products. The first The first steps of the HMS and I-IPPD catalytic reactions are believed to proceed in the same way and lead to an Fe(IV)=O-hydroxyphenylacetate (HPA) intermediate. Further, down the, catalytic cycles, HMS uses Fe(IV)=O to perform hydroxylation of the benzylic carbon, Whereas in HPPD, the reactive oxoferryl intermediate attacks the aromatic ring of HPA. This study focuses on this part of the HMS catalytic cycle that starts from the oxoferryl intermediate and aims to identify interactions within the active site that are responsible for enzyme specificity. To this end, a HMS-Fe(IV)=O-HPA complex was modeled with molecular dynamics simulations On the basis. of the molecular: dynamics. equilibrated structure, active site model suitable for quantum chemical Investigations was constructed and used for density functional theory. (B3LYP) Calculations of the mechanism of the native reaction of HMS, i.e., benzylic hydroxylation, and the alternative electrophilic attack on the ring, which is a step Of the HPPD catalytic cycle: The most important, result of this study is the finding that the conformation of the Ser201 side chain in the second coordination shell has a key role in directing the of Fe(IV)=O. into either the HMS or the HPPD channel

  • 1071. Wormald, P
    et al.
    Lundgren, Bo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Andersson, K
    DePierre, J W
    Interaction of phospholipids with rhodamine 6G in toluene.1992Ingår i: Acta Chemica Scandinavica, ISSN 0904-213X, E-ISSN 1902-3103, Vol. 46, nr 1Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Upon interaction of various glycerophospholipids with Rhodamine 6G in toluene, a typical difference spectrum with an absorption maximum at approximately 515 nm is obtained . This spectrum is obtained with phosphatidylcholine only after treatment with NaCl, which presumably weakens intra- and/or inter-molecular electrostatic binding between the negatively charged phosphate moiety and the protonated nitrogen in this molecule. Absorption at 515 nm was linear for all of the phospholipids investigated from a concentration of approximately 1.2 microM up to at least 50 microM. The highest extinction coefficient was obtained for diphosphatidylglycerol (251 mM-1 cm-1) and all of the compounds tested, with the exception of phosphatidylethanolamine, demonstrated extinction coefficients higher than that of palmitic acid. Thus, the absorption spectrum which results from the interaction of purified glycerophospholipids with Rhodamine 6G in organic solvent is a sensitive measure of the amount of phospholipid present.

  • 1072.
    Wärmländer, Sebastian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Tiiman, Ann
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Tallinn Technical University, Estonia.
    Abelein, Axel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Luo, Jinghui
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Leiden University, Netherlands.
    Söderberg, Kajsa L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biophysical Studies of the Amyloid beta-Peptide: Interactions with Metal Ions and Small Molecules2013Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 14, nr 14, s. 1692-1704Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alzheimer's disease is the most common of the protein misfolding (amyloid) diseases. The deposits in the brains of afflicted patients contain as a major fraction an aggregated insoluble form of the so-called amyloid beta-peptides (A beta peptides): fragments of the amyloid precursor protein of 39-43 residues in length. This review focuses on biophysical studies of the A beta peptides: that is, of the aggregation pathways and intermediates observed during aggregation, of the molecular structures observed along these pathways, and of the interactions of A beta with Cu and Zn ions and with small molecules that modify the aggregation pathways. Particular emphasis is placed on studies based on high-resolution and solid-state NMR methods. Theoretical studies relating to the interactions are also included. An emerging picture is that of A beta peptides in aqueous solution undergoing hydrophobic collapse together with identical partners. There then follows a relatively slow process leading to more ordered secondary and tertiary (quaternary) structures in the growing aggregates. These aggregates eventually assemble into elongated fibrils visible by electron microscopy. Small molecules or metal ions that interfere with the aggregation processes give rise to a variety of aggregation products that may be studied in vitro and considered in relation to observations in cell cultures or in vivo. Although the heterogeneous nature of the processes makes detailed structural studies difficult, knowledge and understanding of the underlying physical chemistry might provide a basis for future therapeutic strategies against the disease. A final part of the review deals with the interactions that may occur between the A beta peptides and the prion protein, where the latter is involved in other protein misfolding diseases.

  • 1073.
    Xia, Zhenlei
    Stockholms universitet.
    In vitro studies on the potential immunosuppressive and antitumor effects of antidepressants: induction of apoptosis in human T-cells and a myeloid leukemia cell line1999Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Antidepressants are widely used in treating depressive disorders. It has been demonstrated that several components of the immune system may be activated in the acute clinical stage of major depression and that such activation may play a role in the onset of depressive symptoms. This suggested involvement of activation of the immune system in depression gives rise to the hypothesis that antidepressants have an inhibitory effect on the immune system.

    Therefore, we were interested in investigating possible molecular effects of antidepressants on cells involved in immunological defenses. We found that incubation of human peripheral T-cells with the antidepressants imipramine, clomipramine and citalopram in vitro inhibits phytohaemagglutinin-induced interlukin-2 (IL-2) and interferon-g(IFN-g) release. The secretion of interlukin-1b(IL-1b), tumor necrosis factor-a(TNF-a) and interlukin-6 (IL-6) by peripheral monocytes caused by lipopolysaccharide is also inhibited by these compounds.

    Our further investigations demonstrated that these drugs induce apoptotic cell death in human peripheral T-cells in vitro , which may account for their immunosuppressive effects. This apoptosis was shown to be dose- and time-dependent and could be abolished by exogenous IL-2 (a pro-inflammatory cytokine), reduced glutathione (an antioxidant) and aurintricarboxylic acid (a nuclease inhibitor). This process could not be inhibited by the translation inhibitor cycloheximide or the transcription inhibitor actinomycin D, indicating that protein and RNA synthesis are not required.

    Furthermore, we have shown that these drugs also induce apoptosis in human acute myeloid leukemia HL-60 cells, which might explain the antineoplastic effects known to be exerted by these compounds. Antidepressant-induced apoptosis in HL-60 cells is associated with an early increase in the production of reactive oxygen species (ROS), subsequent loss of mitochondial membrane potential (Dym) and activation of a caspase-3-like protease(s). Early hypergeneration of ROS may thus be an initial signal in antidepressant-induced apoptosis. Overexpression of the antiapoptotic protein Bcl-2 or Bcl-XL prevents drug-induced apoptosis, as well as loss of Dym, but does not affect the generation of ROS.

  • 1074. Xie, Peiwu
    et al.
    Tu, Tieyao
    Razafimandimbison, Sylvain G.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik. Stockholms universitet, Naturvetenskapliga fakulteten, Bergianska botaniska trädgården (tills m Kungl. Vetenskapsakademien).
    Zhu, Chengjie
    Zhang, Dianxiang
    Phylogenetic position of Guihaiothamnus (Rubiaceae): Its evolutionary and ecological implications2014Ingår i: Molecular Phylogenetics and Evolution, ISSN 1055-7903, E-ISSN 1095-9513, Vol. 78, s. 375-385Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Guihaiothamnus (Rubiaceae) is an enigmatic, monotypic genus endemic to southwestern China. Its generic status has never been doubted because it is morphologically unique by having rosette habit, showy, long-corolla-tubed flowers, and multi-seeded indehiscent berry-like fruits. The genus has been postulated to be a relict in the broad-leaved forests of China, and to be related to the genus Wendlandia, which was placed in the subfamily Cinchonoideae and recently classified in the tribe Augusteae of the subfamily Dialypetalanthoideae. Using combined evidence from palynology, cytology, and DNA sequences of nuclear ITS and four plastid markers (rps16, trnT-F, ndhF, rbcL), we assessed the phylogenetic position of Guihaiothamnus in Rubiaceae. Our molecular phylogenetic analyses placed the genus deeply nested within Wendlandia. This relationship is corroborated by evidence from palynology and cytology. Using a relaxed molecular clock method based on five fossil records, we dated the stem age of Wendlandia to be 17.46 my and, the split between G. acaulis and related Wendlandia species in southwestern China to be 2.11 mya. This young age, coupled with the derived position in Wendlandia, suggests an evolutionary derivation rather than an evolutionary relict of G. acaulis. Its rosette habit and large showy flowers, which are very distinctive from other Wendlandias, are interpreted as a result of recent rapid adaptation to rock and cliff habitats.

  • 1075.
    Yang, Dan-Hui
    Stockholms universitet.
    On the molecular mechanism of LHCII proteolysis during photoacclimation of photosystem II2000Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Higher plants and green algae have a remarkable ability to respond to fluctuations in the light intensity. Plants adapted to low or high light regimes possess larger or smaller light-harvesting antennae respectively. Although this long-term photoacclimation of the PSII antennae is an important factor for the photoprotection of photosynthetic apparatus and it has been extensively described at the physiological level, the underlying molecular mechanisms have remained largely unknown.

    In this thesis, I present new experimental data and a model for the mechanism of LHCII proteolysis during photoacclimation of PSII based on both in vivo and in vitro studies performed in Spinacia oleracea plants. I demonstrate that an endogenous ATP-dependent proteolytic activity is responsible for the acclimative degradation of LHCII in response to increased irradiance thereby reducing the antenna size of PSII. The proteolytic process is tightly regulated at both the enzymatic and substrate levels. Investigations on the substrate regulation were performed using both biochemical and molecular biological approaches, where isolated native and a series of reconstituted recombinant LHCII were employed as substrates. These studies demonstrate a complex multi-step regulation on the substrate level involving protein phosphorylation induced changes in the oligomeric state of LHCII and its subsequent migration from the appressed to the nonappressed thylakoid regions, dephosphorylation of LHCII and its recognition by the membrane associated proteolytic system. It is shown that these steps are controlled directly or indirectly via the N-terminal domain of LHCII.

    Furthermore, the proteolytic system involved has been characterized in vitro and found not to correspond to any previously characterized thylakoid proteases. Purification of the novel proteolytic system from acclimating spinach leaves reveal that it is represented by a 44 kDa protease, probably a chloroplast homologue of the Lon protease in E. coli., which may consist of an ATPase/SSD domain and a protease domain. The fragmentation pattern during LHCII degradation in vitro gives support on requirement of multiple proteolytic cleavages for the complete digestion of LHCII. Finally, the mechanism of redox controlled turnover of LHCII during photoacclimation was studied using Lemna perpusilla wild type and a cytochrome b6f-deficient mutant as a model system. The results strongly suggest that plastoquinone, as an important redox regulator in signal transduction, affects both synthesis and degradation of LHCII during photoacclimation of PSII.

  • 1076. Yang, Qian
    Modulation of adaptive immune responses in mice upon exposure to peroxisome proliferators2001Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    At present, there is an increasing awareness that exposure of mammals to drugs or other foreign chemicals can result in modulation of immunological responses via direct or indirect mechanisms. Changes in the immune system can obviously have profound physiological and pathological consequences. One important family of xenobiotics, the peroxisome proliferators (PPs), including numerous industrial chemicals (e.g., phthalates and perfluoro fatty acids), agrochemicals (e.g., phenoxyacetic acids) and important clinical drugs (e.g., hypolipidemic agents, such as fibrate derivatives, and acetylsalicylic acid) have not yet been examined in this respect.

    Accordingly, examination of possible effects of PPs on the immune system of mice was undertaken in the present study. For the first time, we have demonstrated that PPs do indeed modulate the immune system of mice in a number of ways. (1) Severe thymic and splenic atrophy occur upon treatment of mice with potent PPs. (2) These changes reflect dramatic decreases in the numbers of thymocytes and splenocytes as a consequence of inhibition of thymocyte proliferation. (3) These effects are dose- and time-dependent and are rapidly reversed upon withdrawal of PP from the diet. (4) PP treatment also results in dramatic suppression of the ability of splenocytes to produce specific antibodies in response to T-cell dependent antigens, as well as reducing the proliferation of both T- and B-cells in response to specific activators.

    Further investigation established that an indirect mechanism is involved in these changes. In addition, the atrophy of the thymus and spleen occurred later than hepatic peroxisomal proliferation and required a higher dose, suggesting that these two responses are not coordinated. Finally, by employing transgenic mice, we demonstrated that these immunomodulating effects of PPs are, to a large extent, mediated by the peroxisome proliferator-activated receptor-a (PPARa).

  • 1077. Yazdi, Samira
    et al.
    Stein, Matthias
    Elinder, Fredrik
    Andersson, Magnus
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    The Molecular Basis of Polyunsaturated Fatty Acid Interactions with the Shaker Voltage-Gated Potassium Channel2016Ingår i: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 12, nr 1, artikel-id e1004704Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Voltage-gated potassium (K-V) channels are membrane proteins that respond to changes in membrane potential by enabling K+ ion flux across the membrane. Polyunsaturated fatty acids (PUFAs) induce channel opening by modulating the voltage-sensitivity, which can provide effective treatment against refractory epilepsy by means of a ketogenic diet. While PUFAs have been reported to influence the gating mechanism by electrostatic interactions to the voltage-sensor domain (VSD), the exact PUFA-protein interactions are still elusive. In this study, we report on the interactions between the Shaker K-V channel in open and closed states and a PUFA-enriched lipid bilayer using microsecond molecular dynamics simulations. We determined a putative PUFA binding site in the open state of the channel located at the protein-lipid interface in the vicinity of the extracellular halves of the S3 and S4 helices of the VSD. In particular, the lipophilic PUFA tail covered a wide range of non-specific hydrophobic interactions in the hydrophobic central core of the protein-lipid interface, while the carboxylic head group displayed more specific interactions to polar/charged residues at the extracellular regions of the S3 and S4 helices, encompassing the S3-S4 linker. Moreover, by studying the interactions between saturated fatty acids (SFA) and the Shaker K-V channel, our study confirmed an increased conformational flexibility in the polyunsaturated carbon tails compared to saturated carbon chains, which may explain the specificity of PUFA action on channel proteins.

  • 1078.
    Ye, Weihua
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Spånning, Erika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Interaction of the dual targeting peptide of Thr-tRNA synthetase with the chloroplastic receptor Toc34 in Arabidopsis thaliana2015Ingår i: FEBS Open Bio, E-ISSN 2211-5463, Vol. 5, s. 405-412Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Organellar proteins synthesized in the cytosol are usually selective for only one destination in a cell but some proteins are localized in more than one compartment, for example in both mitochondria and chloroplasts. The mechanism of dual targeting of proteins to mitochondria and chloroplasts is yet poorly understood. Previously, we observed that the dual targeting peptide of threonyl-tRNA synthetase in Arabidopsis thaliana (AtThrRS-dTP) interacts with the mitochondrial receptor AtTom20 mainly through its N-terminal part. Here we report on the interaction of AtThrRS-dTP with the chloroplastic receptor AtToc34, presenting for the first time the mode of interactions of a dual targeting peptide with both Tom20 and Toc34. By NMR spectroscopy we investigated changes in (15)(N) HSQC spectra of AtThrRS-dTP as a function of AtToc34 concentration. Line broadening shows that the interaction with AtToc34 involves residues along the entire sequence, which is not the case for AtTom20. The N-terminal phi chi chi phi phi motif, which plays an important role in AtTom20 recognition, shows no specificity for AtToc34. These results are supported by import competition studies into both mitochondria and chloroplasts, in which the effect of peptides corresponding to different segments of AtThrRS-dTP on in vitro import of organelle specific proteins was examined. This demonstrates that the N-terminal A2-Y29 segment of AtThrRS-dTP is essential for import into both organelles, while the C-terminal L30-P60 part is important for chloroplastic import efficiency. In conclusion, we have demonstrated that the recognition of the dual targeting peptide of AtThr-tRNA synthetase is different for the mitochondrial and chloroplastic receptors. (C) 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license.

  • 1079.
    Ye, Weihua
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Spånning, Erika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Unnerståle, Sofia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gotthold, David
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana2012Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, nr 19, s. 3738-3748Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form a-helical structure in one region, S6F27, and a very weak beta-strand propensity for Q34Q38. The a-helical structured region has an amphiphilic character and a f??ff motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9V28, overlaps with the amphiphilic region, and the other comprises residues L30Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. Database The backbone chemical shift assignments for ThrRS-dTP(260) have been deposited with the Biological Magnetic Resonance Bank (BMRB) under the accession code 18248 Structured digital abstract ThrRS-dTP and Tom20-4 bind by nuclear magnetic resonance (View interaction)

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  • 1080.
    Yentrapalli, Ramesh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi. Helmholtz Zentrum München .
    Azimzadeh, Omid
    Barjaktarovic, Zarko
    Sarioglu, Hakan
    Wojcik, Andrzej
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Harms-Ringdahl, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Atkinson, Michael J.
    Haghdoost, Siamak
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Tapio, Soile
    Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low-dose rate gamma radiation2013Ingår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, nr 7, s. 1096-1107Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chronic low-dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low-dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation-induced senescence. Cellular proteins were quantified using isotope-coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence-related biological pathways were influenced by radiation, including cytoskeletal organization, cellcell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation-induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low-dose irradiation.

  • 1081.
    Yentrapalli, Ramesh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Helmholtz Zentrum München.
    Azimzadeh, Omid
    Sriharshan, Arundhathi
    Malinowsky, Katharina
    Merl, Juliane
    Wojcik, Andrzej
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Harms-Ringdahl, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Atkinson, Michael J.
    Becker, Karl-Friedrich
    Haghdoost, Siamak
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Tapio, Soile
    The PI3K/Akt/mTOR Pathway Is Implicated in the Premature Senescence of Primary Human Endothelial Cells Exposed to Chronic Radiation2013Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 8, s. e70024-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.

  • 1082.
    Yentrapalli, Venkata ramesh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Chronic low-dose gamma radiation exposure induces premature senescence in human umbilical vein endothelial cellsManuskript (preprint) (Övrigt vetenskapligt)
  • 1083.
    Yentrapalli, Venkata Ramesh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Mechanism behind the development of radiation-induced cardiovascular effects2012Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Epidemiological studies on acutely and chronically exposed individuals indicate that ionising radiation could be a risk factor for cardiovascular diseases. Experimental data regarding radiation-induced cardiovascular late effects are limited and biological mechanisms behind these late effects are still unclear. The estimation of risks concerning low-dose rate and low-dose ionising radiation is still challenging due to lack of experimental evidence.

    The first paper of the thesis describes the radiation-induced protein alterations in cardiac tissue of total body irradiated mice. We use a label-free proteomic approach to identify altered proteins from formalin-fixed paraffin-embedded heart tissue. Comparison between the cardiac proteomes of control and irradiated mice indicates the respiratory chain, lipid metabolism and pyruvate metabolism pathways are affected. We suggest that these biological processes may play a vital role in radiation-induced cardiovascular diseases.

    In the second paper (manuscript) we hypothesized that chronic low-dose rate ionising radiation accelerates premature senescence in endothelial cells and that this may contribute to the radiation-induced cardiovascular diseases. We tested our hypothesis by systematically analysing the growth rate, the number of accumulated senescent cells and the protein expression profiles of the chronically exposed primary human umbilical vein endothelial cells until the cells entered senescence. Decrease in cumulative population doublings and increase in senescent-associated beta-galactosidase stained cells show that chronic exposure to radiation accelerates endothelial cells senescence. Our proteomic results suggest that the cytoskeletal organisation, cell-cell communication and adhesion, inflammation and carbohydrate metabolism were influenced by chronic exposure to radiation. A few deregulated proteins have previously been associated with replicative and stress-induced premature senescence. The results presented here provide new insights on radiation-induced premature senescence in endothelial cells and the changes in protein expression associated with this process.

  • 1084.
    Yousif, Rafat
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum.
    Investigating the influence of water in lysozyme structure and dynamics using FT-IR and XRD2019Självständigt arbete på grundnivå (kandidatexamen), 10 poäng / 15 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Water is “the matrix of life” for its fascinating properties. The well-known simple water molecule consists of one oxygen atom and two hydrogen atoms, covering most of planet earth’ssurface. It is the most studied element in science; however, its properties are still not fully understood. Another essential building block of life is proteins, which manifest naturally in aqueous environments. The protein activity is controlled by the protein folding process that is dependent on the surrounding environment. It is hypothesized that the hydrogen bond network of water plays an important role in the folding process. Here, we investigate the protein lysozyme in liquid water as well as in the crystalline state ice Ih, exploring various temperatures, using FT-IR and XRD. Our main finding is that a transition occurs at approximately T=210 K, indicative of the hypothesised protein dynamic “glass” transitionobserved by previous studies in supercooled water at similar temperatures.

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  • 1085. Ytterberg, A. Jimmy
    et al.
    Zubarev, Roman A.
    Baumgarten, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Post-translational targeting of a recombinant protein promotes its efficient secretion into the E. coli periplasmManuskript (preprint) (Övrigt vetenskapligt)
  • 1086.
    Yu, Simei
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    ATPase dependent and independent roles of Brahma in transcription and pre-mRNA processing2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    SWI/SNF is a chromatin-remodeling complex and Brahma (BRM) is the ATPase subunit of SWI/SNF. BRM regulates transcription by remodeling the nucleosomes at promoter regions. BRM is also associated with RNA and affects pre-mRNA processing together with other SWI/SNF subunits. In this thesis, I will discuss the roles of BRM in both transcription and pre-mRNA processing. In Paper I, we showed that BRM, as well as other SWI/SNF subunits SNR1 and MOR, affects the alternative processing of a subset of pre-mRNAs, as shown by microarray analysis. This observation was validated by RNAi experiments both in Drosophila S2 cells and in vivo. In Paper II, we characterized the trans-splicing of transcripts derived from the mod(mdg4) gene. RNA interference (RNAi) and overexpression experiments revealed that BRM regulates the trans-splicing of mod(mdg4)-RX in an ATPase independent manner. In Paper III, we analyzed the expression of two BRM-target genes identified in Paper I, CG44250 and CG44251. RNAi and overexpression experiments showed that the expression levels of these two genes were affected by BRM in a manner that is independent of its ATPase activity. Transcriptome analysis further proved that BRM affects gene expression both in ATPase dependent and independent manners. In Paper IV, we showed that BRM is present at the 3’-end of two analyzed genes, CG5174 and CG2051. BRM facilitates the recruitment of the cleavage and polyadenylation machinery to the cleavage sites through protein-protein interactions that do not require the ATPase activity of BRM. Morevoer, BRM promotes the cleavage of the CG5174 and CG2051 pre-mRNAs. To sum up, SWI/SNF plays important roles not only in transcription but also in pre-mRNA processing. To regulate transcription, BRM can either act as an ATPase-dependent chromatin remodeler or in a manner that does not involve ATPase activity. Additionally, BRM interacts with RNA-binding proteins to regulate the processing of a subset of pre-mRNAs, and this function of BRM is independent of its chromatin remodeling activity.

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  • 1087.
    Yu, Simei
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Gàñez-Zapater, Antoni
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Jordán-Pla, Antonio
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Rolicka, Anna T.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Östlund Farrants, Ann-Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    A role for SWI/SNF in pre-mRNA 3’-end processingManuskript (preprint) (Övrigt vetenskapligt)
  • 1088.
    Yu, Simei
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Waldholm, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Böhm, Stefanie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Brahma regulates a specific trans-splicing event at the mod(mdg4) locus of Drosophila melanogaster2014Ingår i: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 11, nr 2, s. 134-145Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mod(mdg4) locus of Drosophila melanogaster contains several transcription units encoded on both DNA strands. The mod(mdg4) pre-mRNAs are alternatively spliced, and a very significant fraction of the mature mod(mdg4) mRNAs are formed by trans-splicing. We have studied the transcripts derived from one of the anti-sense regions within the mod(mdg4) locus in order to shed light on the expression of this complex locus. We have characterized the expression of anti-sense mod(mdg4) transcripts in S2 cells, mapped their transcription start sites and cleavage sites, identified and quantified alternatively spliced transcripts, and obtained insight into the regulation of the mod(mdg4) trans-splicing. In a previous study, we had shown that the alternative splicing of some mod(mdg4) transcripts was regulated by Brahma (BRM), the ATPase subunit of the SWI/SNF chromatin-remodeling complex. Here we show, using RNA interference and overexpression of recombinant BRM proteins, that the levels of BRM affect specifically the abundance of a trans-spliced mod(mdg4) mRNA isoform in both S2 cells and larvae. This specific effect on trans-splicing is accompanied by a local increase in the density of RNA polymerase II and by a change in the phosphorylation state of the C-terminal domain of the large subunit of RNA polymerase II. Interestingly, the regulation of the mod(mdg4) splicing by BRM is independent of the ATPase activity of BRM, which suggests that the mechanism by which BRM modulates trans-splicing is independent of its chromatin-remodeling activity.

  • 1089.
    Yu, Simei
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Waldholm, Johan
    Jordán-Pla, Antonio
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Källman, Thomas
    Östlund Farrants, Ann-Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    The ATPase-dependent and ATPase-independent functions of Brahma in transcription regulationManuskript (preprint) (Övrigt vetenskapligt)
  • 1090.
    Yu, Xin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Studies of polyglutamine expanded Ataxin-7 toxicity2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

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  • 1091.
    Zadravec, Damir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för fysiologi.
    Tvrdik, Petr
    Guillou, Hervé
    Haslam, Richard
    Kobayashi, Tsutomu
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Napier, Johnathan A
    Capecchi, Mario R
    Jacobsson, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för fysiologi.
    ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis: a prerequisite for male fertility and sperm maturation in mice2011Ingår i: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 52, nr 2, s. 245-255Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    ELOVL2 is a member of the mammalian microsomal ELOVL fatty acid enzyme family, involved in the elongation of very long-chain fatty acids including PUFAs required for various cellular functions in mammals. Here, we used ELOVL2-ablated (Elovl2(-/-)) mice to show that the PUFAs with 24-30 carbon atoms of the ω-6 family in testis are indispensable for normal sperm formation and fertility in male mice. The lack of Elovl2 was associated with a complete arrest of spermatogenesis, with seminiferous tubules displaying only spermatogonia and primary spermatocytes without further germinal cells. Furthermore, based on acyl-CoA profiling, heterozygous Elovl2(+/-) male mice exhibited haploinsufficiency, with reduced levels of C28:5 and C30:5n-6 PUFAs, which gave rise to impaired formation and function of haploid spermatides. These new insights reveal a novel mechanism involving ELOVL2-derived PUFAs in mammals and previously unrecognized roles for C28 and C30 n-6 PUFAs in male fertility. In accordance with the function suggested for ELOVL2, the Elovl2(-/-) mice show distorted levels of serum C20 and C22 PUFAs from both the n-3 and the n-6 series. However, dietary supplementation with C22:6n-3 could not restore male fertility to Elovl2(+/-) mice, suggesting that the changes in n-6 fatty acid composition seen in the testis of the Elovl2(+/-) mice, cannot be compensated by increased C22:6n-3 content.

  • 1092. Zaid, Ahmed
    Transcriptional regulation of human nuclear encoded mitochondrial genes2001Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Biogenesis of mammalian mitochondria requires the participation of both nuclear and mitochondrial genes. This thesis presents studies that characterize the common features of several nuclear encoded mitochondrial promoter genes and proposes the identity of factors regulating the transcription and responsible for coordinated, constitutive expression of diverse mammalian oxidative phosphorylation (OXPHOS) genes.

    As a model, we chose to study the promoters of four nuclear-encoded OXPHOS genes, all which are constitutively expressed, but, in addition, exhibit different responses to hormone and/or growth-activation. They also represent functionally different OXPHOS complexes. These promoters are from the human cytochrome c1, the mitochondrial transcription factor, mtTFA, the F1-ATPase b-subunit, and the adenine nucleotide translocator isoform 2 (ANT2) genes. The results presented in this thesis can be summarized as follows:

    The General transcription factor Sp1 can activate and repress different mammalian OXPHOS genes. Sp1 binding elements (GC boxes) are present in most, if not all, OXPHOS promoter, which suggest that Sp1 may have a general role in regulating the expression of nuclear encoded mitochondrial genes. (b) Sp1-mediated repression and activation from the ANT2 promoter require the D transactivation domain of Sp1 bound to the Cbox. (c) Sp1-mediated repression from the ANT2 promoter is not due to steric interference with the assembly of the transcription machinery. Sp1 bound to the Cbox, under certain conditions, activates the ANT2 promoter (d) Different Sp family members (Sp2, Sp3 and Sp4) differentially affect transcription of the OXPHOS promoters studied. (e) AP-2 enhances Sp1-dependent transactivation of the ANT2 promoter gene. (f) Sp3, but not Sp4, can repress the ANT2 promoter via the Cbox, suggesting that different members of the Sp family can have different roles when they bind to the ANT2 Cbox. (g) Thyroid hormone activates reporter gene expression driven from the ANT2 and cytochrome c1 promoters, which suggests that thyroid hormone induction of some OXPHOS genes is at the transcriptional level.

  • 1093. Zhang, Lifang
    et al.
    Carlberg, Inger
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Norling, Birgitta
    Deletion of Synechocystis sp PCC 6803 Leader Peptidase LepB1 Affects Photosynthetic Complexes and Respiration2013Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, nr 5, s. 1192-1203Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The cyanobacterium Synechocystis sp. PCC 6803 possesses two leader peptidases, LepB1 (SII0716) and LepB2 (SIr1377), responsible for the processing of signal peptide-containing proteins. Deletion of the gene for LepB1 results in an inability to grow photoautotrophically and an extreme light sensitivity. Here we show, using a combination of Blue Native/SDS-PAGE, Western blotting and iTRAQ analysis, that lack of LepB1 strongly affects the cell's ability to accumulate wild-type levels of both photosystem I (PSI) and cytochrome (Cyt) b(6)f complexes. The impaired assembly of PSI and Cyt b(6)f is considered to be caused by the no or slow processing of the integral subunits PsaF and Cyt f respectively. In particular, PsaF, one of the PSI subunits, was found incorporated into PSI in its unprocessed form, which could influence the assembly and/or stability of PSI. In contrast to these results, we found the amount of assembled photosystem II (PSII) unchanged, despite a slower processing of PsbO. Thus, imbalance in the ratios of PSI and Cyt b(6)f to photosystem II leads to an imbalanced photosynthetic electron flow up- and down-stream of the plastoquinone pool, resulting in the observed light sensitivity of the mutant. We conclude that LepB1 is the natural leader peptidase for PsaF, PsbO, and Cyt f. The maturation of PsbO and Cyt f can be partially performed by LepB2, whereas PsaF processing is completely dependent on LepB1. iTRAQ analysis also revealed a number of indirect effects accompanying the mutation, primarily a strong induction of the CydAB oxidase as well as a significant decrease in phycobiliproteins and chlorophyll/heme biosynthesis enzymes.

  • 1094.
    Zhang, Xuan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oglecka, Kamila
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sandgren, Staffan
    Belting, Mattias
    Esbjorner, Elin K.
    Norden, Bengt
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dual functions of the human antimicrobial peptide LL-37-Target membrane perturbation and host cell cargo delivery2010Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, nr 12, s. 2201-2208Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 remain ill-defined. Here, we have investigated the membrane disruption capacity of LL-37 using large unilamellar vesicles (LUVs) composed of varying mixtures of POPC, POPG and cholesterol to mimic target and host membranes respectively. We show that LL-37 is unable to induce leakage of entrapped calcein from zwitterionic POPC LUVs, whereas leakage from LUVs partially composed of POPG is fast and efficient. In accordance with typical antimicrobial peptide behavior, cholesterol diminished LL-37 induced leakage. By using linear dichroism and flow oriented LUVs, we found that LL-37 orients with the axis of its induced alpha-helix parallel to the membrane surface in POPC:POPG (7:3) LUVs. In the same system, we also observed a time-dependent increase of the parallel alpha-helix LD signal on timescales corresponding to the leakage kinetics. The increased LD may be connected to a peptide translocation step, giving rise to mass balance across the membrane. This could end the leakage process before it is complete, similar to what we have observed. Confocal microscopy studies of eukaryotic cells show that LL-37 is able to mediate the cell delivery of non-covalently linked fluorescent oligonucleotides, in agreement with earlier studies on delivery of plasmid DNA (Sandgren et al., J. Biol. Chem. 279 (2004) 17951). These observations highlight the potential dual functions of LL-37 as an antimicrobial agent against bacterial target cells and a cell-penetrating peptide that can deliver nucleic acids into the host cells.

  • 1095.
    Zhang, Zhe
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Enhancing membrane and secretory protein production yields in Escherichia coli2020Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Proteins fulfill essential functions in living cells. To produce sufficient amounts of a protein is essential to study the structure and function of a protein, or to use it for medical purposes. Escherichia coli is a Gram-negative bacterium that is widely used for recombinant protein production. The aim of my PhD studies was to enhance membrane and secretory protein production yields using E. coli. The T7-based protein production system BL21(DE3)/pET was mainly used in my studies. BL21(DE3) contains a strong IPTG-inducible lacUV5 promoter governing the expression of the t7rnap gene encoding the T7RNAP on its chromosome. The target gene is under control of the T7 promoter on the pET plasmid. T7RNAP specifically recognizes the T7 promoter and transcribes the target gene more efficiently than the bacterial RNAP. Unfortunately, the biogenesis machinery for membrane and secretory proteins is usually saturated by the high protein production intensity when the BL21(DE3)/pET system is induced with IPTG, thereby negatively affecting protein production yields. In the first study, we found that when using the BL21(DE3)/pET system omitting the inducer IPTG improved membrane and secretory protein production yields. In previous studies, Lemo21(DE3) was developed to facilitate the production of membrane and secretory proteins. Lemo21(DE3) contains the pLemo plasmid in which the gene encoding the inhibitor of T7RNAP, T7 lysozyme, is under the control of the rhaBAD promoter. The activity of T7RNAP is regulated by synthesizing different levels of T7 lysozyme by adding different amounts of rhamnose. Thus, the production intensity can be modulated such that the biogenesis machinery of membrane and secretory proteins is not saturated upon IPTG induction. In the second study, we combined the key elements from both the pLemo and pET vectors to create the pReX (Regulated eXpression) plasmid to facilitate the use of helper plasmids encoding e.g., chaperones when it is necessary. In the third study, we used the rhaBAD promoter to direct the production of membrane and secretory proteins in a rhamnose metabolism and active uptake deficient strain. The protein production rate can be truly tuned in this setup. Therefore, the production of membrane and secretory proteins can be enhanced by using the right amount of rhamnose in the culture medium. BL21(DE3) contains the λDE3 prophage that carries the t7rnap gene under the control of the lacUV5 promoter. The λDE3 prophage is thought to be stably inserted into the chromosome, but the lytic cycle of the prophage can still be induced by the SOS response inducing antibiotic mitomycin C in the mitomycin C-based bacteriophage test. In the fourth study, we engineered BL21T7 by deleting in BL21(DE3) lysis related genes from the prophage. BL21T7 has similar recombinant protein production characteristics as its ancestor BL21(DE3).

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  • 1096.
    Zhang, Zhe
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ampah-Korsah, Henry
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Karyolaimos, Alexandros
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    de Gier, Jan-Willem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Construction and characterization of the bacteriophage testable BL21(DE3)-derivative BL21T7Manuskript (preprint) (Övrigt vetenskapligt)
  • 1097.
    Zhou, Shu
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pettersson, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Björck, Markus L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dawitz, Hannah
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    NMR structural analysis of yeast Cox13 reveals its C-terminus in interaction with ATPManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The organization of mitochondrial respiratory chain complexes into supercomplexes is vital to cellular activities. In the yeast Saccharomyces cerevisiae, Cox13 is a conserved peripheral subunit of complex IV (cytochrome c oxidase, CytcO) involved in the assembly of monomeric complex IV into supercomplexes. Here we report the solution NMR structure of a Cox13 dimer in detergent micelles. Each Cox13 monomer has three short flexible helices (SH), corresponding to the disordered regions in its homologous X-ray structure, and the dimer formation is mainly induced by the hydrophobic interaction between the sole transmembrane (TM) helix of each monomer. Furthermore, analysis of chemical shift changes upon addition of ATP reveal positions that are able to bind ATP at the conserved sites of the C-terminus with considerable conformational flexibility. From functional analysis of purified CytcO, we conclude that this ATP interaction is inhibitory of catalytic activity. Our results show the structure of an important subunit of yeast CytcO and provide structure-based insight into its ATP interaction.

  • 1098.
    Zhou, Tuping
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Shu, Nanjiang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Hovmöller, Sven
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    A Novel Method for Accurate One-dimensional Protein Structure Prediction Based on Fragment Matching2010Ingår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 26, nr 4, s. 470-477Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Motivation: The precise prediction of one-dimensional (1D) protein structure as represented by the protein secondary structure and 1D string of discrete state of dihedral angles (i.e. Shape Strings) is a prerequisite for the successful prediction of three-dimensional (3D) structure as well as protein-protein interaction. We have developed a novel 1D structure prediction method, called Frag1D, based on a straightforward fragment matching algorithm and demonstrated its success in the prediction of  three sets of 1D structural alphabets, i.e. the classical three-state secondary structure, three-state Shape Strings and eight-state Shape Strings.

    Results: By exploiting the vast protein sequence and protein structure data available, we have brought secondary structure prediction closer to the expected theoretical limit. When tested by a leave-one-out cross validation on a non-redundant set of PDB cutting at 30% sequence identity containing 5860 protein chains, the overall per-residue accuracy for secondary structure prediction, i.e. Q3 is 82.9%. The overall per-residue accuracy for three-state and eight-state Shape Strings are 85.1% and 71.5% respectively. We have also benchmarked our program with the latest version of PSIPRED for secondary structure prediction and our program predicted 0.3% better in Q3 when tested on 2241 chains with the same training set. For Shape Strings, we compared our method with a recently published method with the same dataset and definition as used by that method. Our program predicted at 2.2% better in accuracy for three-state Shape Strings. By quantitatively investigating the effect of data base size on 1D structure prediction we show that the accuracy increases by about 1% with every doubling of the database size.

  • 1099. Zhu, Haining
    et al.
    Zhao, Jun
    Zhu, Beibei
    Collazo, Joanne
    Gal, Jozsef
    Shi, Ping
    Liu, Li
    Ström, Anna-Lena
    University of Kentucky, USA.
    Lu, Xiaoning
    McCann, Richard O.
    Toborek, Michal
    Kyprianou, Natasha
    EMMPRIN regulates cytoskeleton reorganization and cell adhesion in prostate cancer2011Ingår i: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 72, nr 1, s. 72-81Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment. METHODS: In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) versus malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization. RESULTS: EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression, or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins. CONCLUSIONS: Our results suggest that EMMPRIN regulates cell adhesion, invasion, and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis.

  • 1100. Zuber, Bartek
    et al.
    Rudström, Karin
    Ehrnfelt, Cecilia
    Ahlborg, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Mabtech, Sweden.
    Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-gamma Using Human-Bovine Interferon-gamma Chimeras2016Ingår i: Journal of Interferon and Cytokine Research, ISSN 1079-9907, E-ISSN 1557-7465, Vol. 36, nr 9, s. 542-551Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-. Based on the mAbs' (n=12) ability to simultaneously bind hIFN- in ELISA, 2 epitope clusters with 5mAbs in each were defined; 2mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-, epitopes were identified using 7h/bIFN- chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN- residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN--mediated activation of human cells, in line with the involvement of region A in the IFN- receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes.

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