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  • 1.
    Biverståhl, Henrik
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lind, Jesper
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bodor, Andrea
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K(+) channels2009In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1788, no 9, p. 1976-86Article in journal (Refereed)
    Abstract [en]

    The membrane location of two fragments in two different K(+)-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative "paddle" domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T(1) and (13)C-(1)H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. (2)H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.

  • 2.
    Brzezinski, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Ann-Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, no 6-7, p. 710-23Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase is a multisubunit membrane-bound enzyme, which catalyzes oxidation of four molecules of cytochrome c2+ and reduction of molecular oxygen to water. The electrons are taken from one side of the membrane while the protons are taken from the other side. This topographical arrangement results in a charge separation that is equivalent to moving one positive charge across the membrane for each electron transferred to O2. In this reaction part of the free energy available from O2 reduction is conserved in the form of an electrochemical proton gradient. In addition, part of the free energy is used to pump on average one proton across the membrane per electron transferred to O2. Our understanding of the molecular design of the machinery that couples O2 reduction to proton pumping in oxidases has greatly benefited from studies of so called "uncoupled" structural variants of the oxidases. In these uncoupled oxidases the catalytic O2-reduction reaction may display the same rates as in the wild-type CytcO, yet the electron/proton transfer to O2 is not linked to proton pumping. One striking feature of all uncoupled variants studied to date is that the (apparent) pKa of a Glu residue, located deeply within a proton pathway, is either increased or decreased (from 9.4 in the wild-type oxidase). The altered pKa presumably reflects changes in the local structural environment of the residue and because the Glu residue is found near the catalytic site as well as near a putative exit pathway for pumped protons these changes are presumably important for controlling the rates and trajectories of the proton transfer. In this paper we summarize data obtained from studies of uncoupled structural oxidase variants and present a hypothesis that in quantitative terms offers a link between structural changes, modulation of the apparent pKa and uncoupling of proton pumping from O2 reduction.

  • 3.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Biverståhl, Henrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing2011In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1808, no 1, p. 298-306Article in journal (Refereed)
    Abstract [en]

    Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.

  • 4. Georgieva, Polina
    et al.
    Wu, Qian
    McLeish, Michael J.
    Himo, Fahmi
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    The reaction mechanism of phenylethanolamine N-methyltransferase: A density functional theory study2009In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1794, no 12, p. 1831-1837Article in journal (Refereed)
  • 5.
    Glaser, Elzbieta
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The organellar peptidasome, PreP: a journey from Arabidopsis to Alzheimer's disease2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, no 6-7, p. 1076-1080Article in journal (Refereed)
    Abstract [en]

    The novel peptidasome, called presequence protease, PreP, was originally identified and characterized in Arabidopsis thaliana as a mitochondrial matrix and chloroplast stroma localized metalloprotease. PreP has a function as the organellar peptide clearing protease and is responsible for degrading free targeting peptides and also other unstructured peptides up to 65 amino acid residues that might be toxic to organellar functions. PreP contains an inverted Zn-binding motif and belongs to the pitrilysin protease family. The crystal structure of AtPreP refined at 2.1 A demonstrated a unique totally enclosed large cavity of 10000 A3 that opens and closes in response to peptide binding, revealing a novel catalytic mechanism for proteolysis. Homologues of PreP have been found in yeast and human mitochondria. Interestingly, the human PreP, hPreP, is the protease that is responsible for clearing the human brain mitochondria from the toxic amyloid-beta peptide (Abeta) associated with Alzheimer's disease (AD). Accumulation of Abeta has been shown in the brain mitochondria from AD patients and mutant transgenic mice overexpressing Abeta. Here, we present a review of our present knowledge on structural and functional characteristics of PreP and discuss its mitochondrial Abeta-degrading activity in the human brain mitochondria in relation to AD.

  • 6.
    Holm, Tina
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Räägel, Helin
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hein, Margot
    Mäe, Maarja
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pooga, Margus
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity2011In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1808, no 6, p. 1544-1551Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are a promising group of delivery vectors for various therapeutic agents but their application is often hampered by poor stability in the presence of serum. Different strategies to improve peptide stability have been exploited, one of them being "retro-inversion" (RI) of natural peptides. With this approach the stability of CPPs has been increased, thereby making them more efficient transporters. Several RI-CPPs were here assessed and compared to the corresponding parent peptides in different cell-lines. Surprisingly, treatment of cells with these peptides induced trypsin insensitivity and rapid severe toxicity in contrast to l-peptides. This was measured as reduced metabolic activity and condensed cell nuclei, in parity with the apoptosis inducing agent staurosporine. Furthermore, effects on mitochondrial network, focal adhesions, actin cytoskeleton and caspase-3 activation were analyzed and adverse effects were evident at 20μM peptide concentration within 4h while parent l-peptides had negligible effects. To our knowledge this is the first time RI peptides are reported to cause cellular toxicity, displayed by decreased metabolic activity, morphological changes and induction of apoptosis. Considering the wide range of research areas that involves the use of RI-peptides, this finding is of major importance and needs to be taken under consideration in applications of RI-peptides.

  • 7.
    Huang, Yafei
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reimann, Joachim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Singh, Laila M R
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Substrate binding and the catalytic reactions in cbb3-type oxidases: the lipid membrane modulates ligand binding2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, no 6-7, p. 724-31Article in journal (Refereed)
    Abstract [en]

    Heme-copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme-copper oxidases of the cbb3-type are only found in bacteria, often pathogenic ones since they have a low Km for O2, enabling the bacteria to colonize semi-anoxic environments. Cbb3-type (C) oxidases are highly divergent from the mitochondrial-like aa3-type (A) oxidases, and within the heme-copper oxidase family, cbb3 is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N2O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme-copper oxidases are manifested in the lack in cbb3 of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O2 and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O2 and NO reduction as well as the proton-pumping mechanism in all heme-copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein-lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein-membrane interactions in cbb3 oxidases, and possible physiological consequences for this behavior are discussed.

  • 8.
    Lepp, Håkan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Internal charge transfer in cytochrome c oxidase at a limited proton supply: proton pumping ceases at high pH.2009In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1790, no 6, p. 552-7Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O(2) is linked to proton uptake from solution to form H(2)O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane. METHODS: In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O(2). These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles. RESULTS: The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O(2) reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH. CONCLUSIONS: The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the "wrong" side of the membrane) and that at this pH the net proton-uptake stoichiometry is approximately 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme. GENERAL SIGNIFICANCE: These results provide new insights into the function of cytochrome c oxidase.

  • 9.
    Lind, Jesper
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordin, Jon
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lipid dynamics in fast-tumbling bicelles with varying bilayer thickness: Effect of model transmembrane peptides2008In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1778, no 11, p. 2526-2534Article in journal (Refereed)
    Abstract [en]

    The morphology of q=0.5 fast-tumbling bicelles prepared with three different acyl chain lengths has been investigated by NMR. It is shown that bicelles prepared with DLPC (12 C) and DHPC are on average larger than those containing DMPC or DPPC (14 and 16 C) and DHPC, which may be due to a higher degree of mixing between DLPC and DHPC. The fast internal mobility of the lipids was determined from natural abundance carbon-13 relaxation. A similar dynamical behaviour of the phospholipids in the three different bicelles was observed, although the DPPC lipid acyl chain displayed a somewhat lower degree of mobility, as evidenced by higher generalized order parameters throughout the acyl chain. Carbon-13 relaxation was also used to determine the effect of different model transmembrane peptides, with flanking Lys residues, on the lipid dynamics in the three different bicelles. All peptides had the effect of increasing the order parameters for the DLPC lipid, while no effect was observed on the longer lipid chains. This effect may be explained by a mismatch between the hydrophobic length of the peptides and the DLPC lipid acyl chain.

  • 10. Lundgren, B
    et al.
    Bergstrand, A
    Karlsson, K
    DePierre, J W
    Effects of dietary treatment with clofibrate, nafenopin or WY-14.643 on mitochondria and DNA in mouse liver.1990In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1035, no 2Article in journal (Refereed)
    Abstract [en]

    Male C57bl/6 mice were administered clofibrate (0.5%, w/w), nafenopin (0.125%, w/w) or WY-14.643 (0.125%, w/w) in their diet for 4 days. Assay of eight mitochondrial marker enzymes, -i.e., malate and glutamate dehydrogenases (matrix markers), cytochrome oxidase and cytochromes c + c1 and a (inner membrane), adenylate kinase (intermembrane space) and monoamine oxidase and microsomal glutathione transferase (outer membrane)--and morphometric analysis of electron micrographs was used to examine hepatic mitochondria after treatment with these peroxisome proliferators. A moderate increase in the number of hepatic mitochondrial profiles, with a simultaneous decrease in the average size of these organelles, was observed. The total mitochondrial volume is apparently unchanged during this process. An important experimental consequence of the apparent decrease in mitochondrial size is the redistribution of a large portion of the total hepatic mitochondria from the 'nuclear' to the mitochondrial fraction. A similar effect was seen with rats.

  • 11.
    Mahammad, Saleemulla
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions.2008In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1778, no 5, p. 1251-8Article in journal (Refereed)
    Abstract [en]

    Methyl-beta-cycloclextrin (MBCD) is frequently used to acutely deplete cells of cholesterol. A widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts and that sensitivity to MBCD is proof of lipid raft involvement in a cellular process. To analyse any MBCD preference systematically, progressive cholesterol depletion of Jurkat T cells was performed using MBCD and [H-3]-cholesterol. It was found that at 37 degrees C, MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions and that the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, cells restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, mere incubation at 0 degrees C caused a loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. Moreover, only 35% of total cholesterol could be extracted by MBCD at 0 degrees C and was accompanied by a complete loss of plasma membrane and endocytotic recycling centre filipin staining. This study clearly shows that MBCD does not specifically extract cholesterol from any cellular fraction, that cholesterol redistributes upon temperature changes and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol.

  • 12.
    Mattsson, Charlotte L
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Physiology.
    Andersson, Emma R
    Nedergaard, Jan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Differential involvement of caveolin-1 in brown adipocyte signaling: impaired beta3-adrenergic, but unaffected LPA, PDGF and EGF receptor signaling.2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1803, no 8, p. 983-9Article in journal (Refereed)
    Abstract [en]

    Caveolae and caveolin have been implicated as being involved in the signal transduction of many receptors, including the EGF, PDGF, LPA and beta3-adrenergic receptors. To investigate the role of caveolin-1 (Cav1) in these signaling pathways in brown adipose tissue, primary brown adipocyte cultures from Cav1-ablated mice and wild-type mice were investigated. In pre-adipocytes, Cav1-ablation affected neither the G-protein coupled LPA receptor signaling to Erk1/2, nor the receptor tyrosine kinases PDGF- or EGF-receptor signaling to Erk1/2. Mature primary Cav1-/- brown adipocytes accumulated lipids and expressed aP2 to the same extent as did wild-type cells. However, the cAMP levels induced by the beta3-adrenergic receptor agonist CL316,243 were lower in the Cav1-/- cultures, with an unchanged EC50 for CL316,243. Also the response to the direct adenylyl cyclase agonist forskolin was reduced. Thus, in brown adipocytes, Cav1 is apparently required for an intact response to adenylyl cyclase-linked agonists/activators, whereas other signaling pathways examined function without Cav1

  • 13.
    Mäe, Maarja
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. Tallinn University of Technology, Estonia.
    Myrberg, Helena
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Paves, Heiti
    Valkna, Andres
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Internalisation of cell-penetrating peptides into tobacco protoplasts2005In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1669, no 2, p. 101-107Article in journal (Refereed)
    Abstract [en]

    Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.

  • 14.
    Nørholm, Morten H H
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Light, Sara
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Virkki, Minttu T I
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Daley, Daniel O
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Manipulating the genetic code for membrane protein production: What have we learnt so far?2012In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1818, no 4: S1, p. 1091-1096Article in journal (Refereed)
    Abstract [en]

    With synthetic gene services, molecular cloning is as easy as ordering a pizza. However choosing the right RNA code for efficient protein production is less straightforward, more akin to deciding on the pizza toppings. The possibility to choose synonymous codons in the gene sequence has ignited a discussion that dates back 50years: Does synonymous codon use matter? Recent studies indicate that replacement of particular codons for synonymous codons can improve expression in homologous or heterologous hosts, however it is not always successful. Furthermore it is increasingly apparent that membrane protein biogenesis can be codon-sensitive. Single synonymous codon substitutions can influence mRNA stability, mRNA structure, translational initiation, translational elongation and even protein folding. Synonymous codon substitutions therefore need to be carefully evaluated when membrane proteins are engineered for higher production levels and further studies are needed to fully understand how to select the codons that are optimal for higher production. This article is part of a Special Issue entitled: Protein Folding in Membranes.

  • 15.
    Oglęcka, Kamila
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundberg, Pontus
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Magzoub, Mazin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, L. E. Göran
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects2008In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1778, no 1, p. 206-213Article in journal (Refereed)
    Abstract [en]

    We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23-28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1-28 (mPrPp(1-28)) and 2350 (rnPrPp(23-50)), respectively. In erythrocytes, mPrPp(1-28) induced similar to 60% haemoglobin leakage after 30 min, whereas mprPp(23-50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be similar to 12% for 50 mu M mPrPp(1-28), and similar to 1% for 50 mu M mPrPp(23-50). Circular dichroism spectra showed structure induction of mPrPp(1-28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mprPp(23-50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(I-28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23-50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.

  • 16.
    Palm, Caroline
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jayamanne, Mala
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kjellander, Marcus
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Peptide degradation is a critical determinant for cell-penetrating peptide uptake2007In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1768, no 7, p. 1769-1776Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptide mediated uptake of labels appears to follow an equilibrium-like process. However, this assumption is only valid if the peptides are stabile. Hence, in this study we investigate intracellular and extracellular peptide degradation kinetics of two fluorescein labeled cell-penetrating peptides, namely MAP and penetratin, in Chinese hamster ovarian cells. The degradation and uptake kinetics were assessed by RP-HPLC equipped with a fluorescence detector. We show that MAP and penetratin are rapidly degraded both extracellularly and intracellularly giving rise to several degradation products. Kinetics indicates that intracellularly, the peptides exist in (at least) two distinct pools: one that is immediately degraded and one that is stabile. Moreover, the degradation could be decreased by treating the peptides with BSA and phenanthroline and the uptake was significantly reduced by cytochalasin B, chloroquine and energy depletion. The results indicate that the extracellular degradation determines the intracellular peptide concentration in this system and therefore the stability of cell-penetrating peptides needs to be evaluated.

  • 17.
    Reimann, Joachim
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Flock, Ulrika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lepp, Håkan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Honigmann, Alf
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A pathway for protons in nitric oxide reductase from Paracoccus denitrificans2007In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1767, no 5, p. 362-373Article in journal (Refereed)
    Abstract [en]

    Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N2O (2NO + 2e(-) + 2H(+) -> N2O + H2O) as part of the denitriffication process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O-2-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O-2, we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O-2 resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O-2 shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O-2 as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O-2 show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau = 15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.

  • 18.
    Shabalina, Irina G
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Physiology.
    Hoeks, Joris
    Kramarova, Tatiana V
    Schrauwen, Patrick
    Cannon, Barbara
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Physiology.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Physiology.
    Cold tolerance of UCP1-ablated mice: A skeletal muscle mitochondria switch toward lipid oxidation with marked UCP3 up-regulation not associated with increased basal, fatty acid- or ROS-induced uncoupling or enhanced GDP effects.2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, no 6-7, p. 968-80Article in journal (Refereed)
    Abstract [en]

    Mice lacking the thermogenic mitochondrial membrane protein UCP1 (uncoupling protein 1) - and thus all heat production from brown adipose tissue - can still adapt to a cold environment (4 degrees C) if successively transferred to the cold. The mechanism behind this adaptation has not been clarified. To examine possible adaptive processes in the skeletal muscle, we isolated mitochondria from the hind limb muscles of cold-acclimated wild-type and UCP1(-/-) mice and examined their bioenergetic chracteristics. We observed a switch in metabolism, from carbohydrate towards lipid catabolism, and an increased total mitochondrial complement, with an increased total ATP production capacity. The UCP1(-/-) muscle mitochondria did not display a changed state-4 respiration rate (no uncoupling) and were less sensitive to the uncoupling effect of fatty acids than the wild-type mitochondria. The content of UCP3 was increased 3-4 fold, but despite this, endogenous superoxide could not invoke a higher proton leak, and the small inhibitory effect of GDP was unaltered, indicating that it was not mediated by UCP3. Double mutant mice (UCP1(-/-) plus superoxide dismutase 2-overexpression) were not more cold sensitive than UCP1(-/-), bringing into question an involvement of reactive oxygen species (ROS) in activation of any alternative thermogenic mechanism. We conclude that there is no evidence for an involvement of UCP3 in basal, fatty-acid- or superoxide-stimulated oxygen consumption or in GDP sensitivity. The adaptations observed did not imply any direct alternative process for nonshivering thermogenesis but the adaptations observed would be congruent with adaptation to chronically enhanced muscle activity caused by incessant shivering in these mice.

  • 19.
    Shabalina, Irina G
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Physiology.
    Ost, Mario
    Petrovic, Natasa
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Physiology.
    Vrbacky, Marek
    Nedergaard, Jan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Physiology.
    Cannon, Barbara
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Physiology.
    Uncoupling protein-1 is not leaky.2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, no 6-7, p. 773-84Article in journal (Refereed)
    Abstract [en]

    The activity of uncoupling protein-1 (UCP1) is rate-limiting for nonshivering thermogenesis and diet-induced thermogenesis. Characteristically, this activity is inhibited by GDP experimentally and presumably mainly by cytosolic ATP within brown-fat cells. The issue as to whether UCP1 has a residual proton conductance even when fully saturated with GDP/ATP (as has recently been suggested) has not only scientific but also applied interest, since a residual proton conductance would make overexpressed UCP1 weight-reducing even without physiological/pharmacological activation. To examine this question, we have here established optimal conditions for studying the bioenergetics of wild-type and UCP1(-/-) brown-fat mitochondria, analysing UCP1-mediated differences in parallel preparations of brown-fat mitochondria from both genotypes. Comparing different substrates, we find that pyruvate (or palmitoyl-l-carnitine) shows the largest relative coupling by GDP. Comparing albumin concentrations, we find the range 0.1-0.6% optimal; higher concentrations are inhibitory. Comparing basic medium composition, we find 125mM sucrose optimal; an ionic medium (50-100mM KCl) functions for wild-type but is detrimental for UCP1(-/-) mitochondria. Using optimal conditions, we find no evidence for a residual proton conductance (not a higher post-GDP respiration, a lower membrane potential or an altered proton leak at highest common potential) with either pyruvate or glycerol-3-phosphate as substrates, nor by a 3-4-fold alteration of the amount of UCP1. We could demonstrate that certain experimental conditions, due to respiratoty inhibition, could lead to the suggestion that UCP1 possesses a residual proton conductance but find that under optimal conditions our experiments concur with implications from physiological observations that in the presence of inhibitory nucleotides, UCP1 is not leaky.

  • 20. Shi, Ping
    et al.
    Ström, Anna-Lena
    University of Kentucky, USA.
    Gal, Jozsef
    Zhu, Haining
    Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1802, no 9, p. 707-716Article in journal (Refereed)
    Abstract [en]

    Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper-zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interacted and colocalized with the retrograde dynein-dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early presymptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the slowing of anterograde transport of dynein heavy chain as a cargo was observed in the presymptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members, which, in turn, could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affects different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which, subsequently, contribute to the propagation of toxic effects and ultimately motor neuron death in ALS.

  • 21.
    Vasconcelos, Luís
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Madani, Fatemeh
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Arukuusk, Piret
    Pärnaste, Ly
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Effects of cargo molecules on membrane perturbation caused by transportan10 based cell-penetrating peptides2014In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1838, no 12, p. 3118-3129Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides with the ability to escape endosomes and reach the target are of great value as delivery vectors for different bioactive cargoes and future treatment of human diseases. We have studied two such peptides, NickFect1 and NickFect51, both originated from stearylated transportan10 (PF3). To obtain more insight into the mechanism(s) of peptide delivery and the biophysical properties of an efficient vector system, we investigated the effect of different bioactive oligonucleotide cargoes on peptide-membrane perturbation and peptide structural induction. We studied the membrane interactions of the peptides with large unilamellar vesicles and compared their effects with parent peptides transportan10 and PF3. In addition, cellular uptake and peptide-mediated oligonucleotide delivery were analyzed. Calcein leakage experiments showed that similar to transportan10, NickFect51 caused a significant degree of membrane leakage, whereas NickFect1, similar to PF3, was less membrane perturbing. The results are in agreement with previously published results indicating that NickFect51 is a more efficient endosomal escaper. However, the presence of a large cargo like plasmid DNA inhibited NickFect's membrane perturbation and cellular uptake efficiency of the peptide was reduced. We conclude that the pathway for cellular uptake of peptide complexes is cargo dependent, whereas the endosomal escape efficacy depends on peptide hydrophobicity and chemical structure. For small interfering RNA delivery, NickFect51 appears to be optimal. The biophysical signature shows that the peptide alone causes membrane perturbation, but the cargo complex does not. These two biophysical characteristics of the peptide and its cargo complex may be the signature of an efficient delivery vector system.

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