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  • 101. Nakase, Ikuhiko
    et al.
    Akita, Hidetaka
    Kogure, Kentaro
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Harashima, Hideyoshi
    Futaki, Shiroh
    Efficient Intracellular Delivery of Nucleic Acid Pharmaceuticals Using Cell-Penetrating Peptides2012Ingår i: Accounts of Chemical Research, ISSN 0001-4842, E-ISSN 1520-4898, Vol. 45, nr 7, s. 1132-1139Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Over the last 20 years, researchers have designed or discovered peptides that can permeate membranes and deliver exogenous molecules inside a cell. These peptides, known as cell-penetrating peptides (CPPs), typically consist of 6-30 residues, including HIV TAT peptide, penetratin, oligoarginine, transportan, and TP10. Through chemical conjugation or noncovalent complex formation, these structures successfully deliver bioactive and membrane-impermeable molecules into cells. CPPs have also gained attention as an attractive vehicle for the delivery of nucleic add pharmaceuticals (NAPs), including genes/plasmids, short oligonucleotides, and small interference RNAs and their analogues, due to their high internalization efficacy, low cytotoxicity, and flexible structural design. In this Account, we survey the potential of CPPs for the design and optimization of NAP delivery systems. First, we describe the impact of the N-terminal stearylation of CPPs. Endocytic pathways make a major contribution to the cellular uptake of NAPS. Stearylation at the N-terminus of CPPs with stearyl-octaarginine (R8), stearyl-(RxR)(4), and stearyl-TP10 prompts the formation of a self-assembled core shell nanoparticle with NAPS, a compact structure that promotes cellular uptake. Researchers have designed modifications such as the addition of trifluoromethylquinoline moieties to lysine residues to destabilize endosomes, as exemplified by PepFect 6, and these changes further improve biological responsiveness. Alternatively, stearylation also allows implantation of CPPs onto the surface of liposomes. This feature facilitates programmed packaging to establish multifunctional envelope-type nanodevices (MEND). The R8-MEND showed high transfection efficiency comparable to that of adenovirus in non-dividing cells. Understanding the cellular uptake mechanisms of CPPs will further improve CPP-mediated NAP delivery. The cellular uptake of CPPs and their NAP complex involves various types of endocytosis. Macropinocytosis, a mechanism which is also activated in response to stimuli such as growth factors or viruses, is a primary pathway for arginine-rich CPPs because high cationic charge density promotes this endocytic pathway. The use of larger endosomes (known as macropinosomes) rather than clathrin- or caveolae-mediated endocytosis has been reported in macropinocytosis which would also facilitate the endocytosis of NAP nanoparticles into cells.

  • 102.
    Oglęcka, Kamila
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Magzoub, Mazin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Eriksson, L. E. Göran
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Relevance of the N-terminal NLS-like sequence for membrane interactions of the Prion protein2007Ingår i: Biochimica et Biophysica Acta. MR. Reviews on Biomembranes, ISSN 0304-4157, E-ISSN 1879-257X, Vol. 1778, nr 1, s. 206-213Artikel i tidskrift (Refereegranskat)
    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 23–50 (mPrPp(23–50)), respectively. In erythrocytes, mPrPp(1–28) induced 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 12% for 50 μM mPrPp(1–28), and 1% for 50 μM 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(1–28) 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.

  • 103.
    Oglęcka, Kamila
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Magzoub, Mazin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Eriksson, L. E. Göran
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects2008Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1778, nr 1, s. 206-213Artikel i tidskrift (Refereegranskat)
    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.

  • 104. Owen, Michael C.
    et al.
    Gnutt, David
    Gao, Mimi
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Winter, Roland
    Ebbinghaus, Simon
    Strodel, Birgit
    Effects of in vivo conditions on amyloid aggregation2019Ingår i: Chemical Society Reviews, ISSN 0306-0012, E-ISSN 1460-4744, Vol. 48, nr 14, s. 3946-3996Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    One of the grand challenges of biophysical chemistry is to understand the principles that govern protein misfolding and aggregation, which is a highly complex process that is sensitive to initial conditions, operates on a huge range of length- and timescales, and has products that range from protein dimers to macroscopic amyloid fibrils. Aberrant aggregation is associated with more than 25 diseases, which include Alzheimer's, Parkinson's, Huntington's, and type II diabetes. Amyloid aggregation has been extensively studied in the test tube, therefore under conditions that are far from physiological relevance. Hence, there is dire need to extend these investigations to in vivo conditions where amyloid formation is affected by a myriad of biochemical interactions. As a hallmark of neurodegenerative diseases, these interactions need to be understood in detail to develop novel therapeutic interventions, as millions of people globally suffer from neurodegenerative disorders and type II diabetes. The aim of this review is to document the progress in the research on amyloid formation from a physicochemical perspective with a special focus on the physiological factors influencing the aggregation of the amyloid-beta peptide, the islet amyloid polypeptide, alpha-synuclein, and the hungingtin protein.

  • 105. Pansieri, Jonathan
    et al.
    Ostojic, Lucija
    Iashchishyn, Igor A.
    Magzoub, Mazin
    Wallin, Cecilia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mai, Nguyen
    Smirnovas, Vytautas
    Svedruzic, Zeljko
    Morozova-Roche, Ludmilla A.
    Pro-Inflammatory S100A9 Protein Aggregation Promoted by NCAM1 Peptide Constructs2019Ingår i: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 14, nr 7, s. 1410-1417Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Amyloid cascade and neuroinflammation are hallmarks of neurodegenerative diseases, and pro-inflammatory S100A9 protein is central to both of them. Here, we have shown that NCAM1 peptide constructs carrying polycationic sequences derived from A beta peptide (KKLVFF) and PrP protein (KKRPKP) significantly promote the S100A9 amyloid self-assembly in a concentration-dependent manner by making transient interactions with individual S100A9 molecules, perturbing its native structure and acting as catalysts. Since the individual molecule misfolding is a rate-limiting step in S100A9 amyloid aggregation, the effects of the NCAM1 construct on the native S100A9 are so critical for its amyloid self-assembly. S100A9 rapid self assembly into large aggregated clumps may prevent its amyloid tissue propagation, and by modulating S100A9 aggregation as a part of the amyloid cascade, the whole process may be effectively tuned.

  • 106.
    Papadopoulos, E
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oglecka, K
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, L
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, J
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wright, PE
    Dyson, HJ
    Gräslund, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    NMR solution structure of the peptide fragment 1-30, derived from unprocessed mouse Doppel protein, in DHPC micelles.2006Ingår i: Biochemistry, ISSN 0006-2960, Vol. 45, nr 1, s. 159-66Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The downstream prion-like Doppel (Dpl) protein is a homologue related to the prion protein (PrP). Dpl is expressed in the brains of mice that do not express PrP, and Dpl is known to be toxic to neurons. One mode of toxicity has been suggested to involve direct membrane interactions. PrP under certain conditions of cell trafficking retains an uncleaved signal peptide, which may also hold for the much less studied Dpl. For a peptide with a sequence derived from the N-terminal part (1-30) of mouse Dpl (mDpl(1-30)) CD spectroscopy shows about 40% alpha-helical structure in DHPC and SDS micelles. In aqueous solution it is mostly a random coil. The three-dimensional solution structure was determined by NMR for mDpl(1-30) associated with DHPC micelles. 2D 1H NMR spectra of the peptide in q = 0.25 DMPC/DHPC bicelles only showed signals from the unstructured termini, indicating that the structured part of the peptide resides within the lipid bilayer. Together with 2H2O exchange data in the DHPC micelle solvent, these results show an alpha-helix protected from solvent exchange between residues 7 and 19, and suggest that the alpha-helical segment can adopt a transmembrane localization also in a membrane. Leakage studies with entrapped calcein in large unilamellar phospholipid vesicles showed that the peptide is almost as membrane perturbing as melittin, known to form pores in membranes. The results suggest a possible channel formation mechanism for the unprocessed Dpl protein, which may be related to toxicity through direct cell membrane interaction and damage.

  • 107.
    Papadopoulos, Evangelos
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Collet, Jean-Francois
    Vukojevic, Vladana
    Billeter, Martin
    Holmgren, Arne
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vlamis-Gardikas, Alexios
    Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli2012Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1824, nr 12, s. 1401-1408Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The gene ygiT (mqsA) of Escherichia coli encodes MqsA, the antitoxin of the motility quorum sensing regulator (MqsR). Both proteins are considered to form a DNA binding complex and to be involved in the formation of biofilms and persisters. We have determined the three-dimensional solution structure of MqsA by high-resolution NMR. The protein comprises a well-defined N-terminal domain with a Zn finger motif usually found in eukaryotes, and a defined C-terminal domain with a typical prokaryotic DNA binding helix-turn-helix motif. The two well-defined domains of MqsA have almost identical structure in solution and in the two published crystal structures of dimeric MqsA bound to either MqsR or DNA. However, the connection of the two domains with a flexible linker yields a large variety of possible conformations in solution, which is not reflected in the crystal structures. MqsA binds Zn with all four cysteines, a stoichiometry of 1:1 and a femtomolar affinity (K-a >= 10(17) M-1 at 23 degrees C, pH 7.0).

  • 108.
    Papapopoulos, Evangelos
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Billeter, Martin
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vlamis-Gardikas, Alexios
    Assignment of 1H, 13C, and 15N resonances of YgiT, a putative DNA interacting protein from E. coli, containing one HTH and two CxxC motifs2007Ingår i: Biomol. NMR Assign., ISSN 1874-2718 (Print) 1874-270X (Online), Vol. 1, s. 217-219Artikel i tidskrift (Refereegranskat)
  • 109.
    Paul, Suman
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jenistova, Adela
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vosough, Faraz
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berntsson, Elina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mörman, Cecilia
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian K. T. S.
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.
    Barth, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    13C- and 15N-labeling of amyloid-β and inhibitory peptides to study their interaction via nanoscale infrared spectroscopy2023Ingår i: Communications Chemistry, E-ISSN 2399-3669, Vol. 6, nr 1, artikel-id 163Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Interactions between molecules are fundamental in biology. They occur also between amyloidogenic peptides or proteins that are associated with different amyloid diseases, which makes it important to study the mutual influence of two polypeptides on each other's properties in mixed samples. However, addressing this research question with imaging techniques faces the challenge to distinguish different polypeptides without adding artificial probes for detection. Here, we show that nanoscale infrared spectroscopy in combination with C-13, N-15-labeling solves this problem. We studied aggregated amyloid-& beta; peptide (A & beta;) and its interaction with an inhibitory peptide (NCAM1-PrP) using scattering-type scanning near-field optical microscopy. Although having similar secondary structure, labeled and unlabeled peptides could be distinguished by comparing optical phase images taken at wavenumbers characteristic for either the labeled or the unlabeled peptide. NCAM1-PrP seems to be able to associate with or to dissolve existing A & beta; fibrils because pure A & beta; fibrils were not detected after mixing. Interactions of proteins or polypeptides with different secondary structures can be studied in a mixture by nanoscale infrared spectroscopy, however, this technique remains challenging for polypeptides with similar secondary structures. Here, the authors demonstrate clear discrimination of two polypeptides from a mixture by scattering-type scanning near-field optical microscopy when one of the components is labeled with C-13- and N-15-isotopes.

  • 110.
    Peralvarez-Marin, Alex
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mateos, Laura
    Zhang, Ce
    Singh, Shalini
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cedazo-Minguez, Angel
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Morozova-Roche, Ludmilla
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Barth, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Influence of Residue 22 on the Folding, Aggregation Profile, and Toxicity of the Alzheimer's Amyloid beta Peptide2009Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 97, nr 1, s. 277-285Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several biophysical techniques have been used to determine differences in the aggregation profile (i.e., the secondary structure, aggregation propensity, dynamics, and morphology of amyloid structures) and the effects on cell viability of three variants of the amyloid beta peptide involved in Alzheimer's disease. We focused our study on the Glu(22) residue, comparing the effects of freshly prepared samples and samples aged for at least 20 days. In the aged samples, a high propensity for aggregation and beta-sheet secondary structure appears when residue 22 is capable of establishing polar (Glu(22) in wild-type) or hydrophobic (Val(22) in E22V) interactions. The Arctic variant (E22G) presents a mixture of mostly disordered and a-helix structures (with low beta-sheet contribution). Analysis of transmission electron micrographs and atomic force microscopy images of the peptide variants after aging showed significant quantitative and qualitative differences in the morphology of the formed aggregates. The effect on human neuroblastoma cells of these A beta(12-28) variants does not correlate with the amount of beta-sheet of the aggregates. In samples allowed to age, the native sequence was found to have an insignificant effect on cell viability, whereas the Arctic variant (E22G), the E22V variant, and the slightly-aggregating control (F19G-F20G) had more prominent effects.

  • 111.
    Popović-Bijelić, Ana
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kowol, Christian R.
    Lind, Maria E. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Luo, Jinghui
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Himo, Fahmi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Enyedy, Éva A.
    Arion, Vladimir B.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): A combined experimental and theoretical study2011Ingår i: European Journal of Inorganic Chemistry, ISSN 1434-1948, E-ISSN 1099-1948, Vol. 105, nr 11, s. 1422-1431Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper(II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)–Triapine are reduced to the iron(II)–Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron(II)–Triapine complex are formed. Formation of the iron(II)–Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical.

  • 112.
    Popović-Bijelić, Ana
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Voevodskaya, Nina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Domkin, Vladimir
    Thelander, Lars
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Metal binding and activity of ribonucleotide reductase protein R2 mutants: Conditions for formation of the mixed manganese-iron cofactor2009Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 27, s. 6532-6539Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (C. tm.) lacks the tyrosyl radical and uses a Mn(IV)-Fe(III) cluster for cysteinyl radical initiation in the large subunit. Here we investigated and compared the metal content and specific activity of the C. tm. wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli. Our results indicate that the affinity of the RNR R2 proteins for binding metals is determined by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins. In mouse R2, this tyrosyl residue is crucial for the activity of the enzyme, but in C. tm., the corresponding phenylalanine plays no obvious role in activation or catalysis. However, for the C. tm. wild-type R2 protein to bind Mn and gain high specific activity, there seems to be a strong preference for F over Y at this position. In studies of mouse RNR, we find that the native R2 protein does not bind Mn whereas its Y177F mutant incorporates a significant amount of Mn and exhibits 1.4% of native mouse RNR activity. The observation suggests that a manganese-iron cofactor is associated with the weak activity in this protein.

  • 113. Rajkovic, Andrei
    et al.
    Kanchugal, Sandesh
    Abdurakhmanov, Eldar
    Howard, Rebecca
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Erwin, Joseph
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Barrera Saldaña, Hugo A.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielson, Helena
    Coulbourn Flores, Samuel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Swedish University of Agricultural Sciences, Sweden.
    Amino acid substitutions in human growth hormone affect coiled-coil content and receptor binding2023Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 18, nr 3, artikel-id e0282741Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH.

  • 114.
    Riboni, Nicolò
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi. University of Parma, Italy.
    Quaranta, Alessandro
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Motwani, Hitesh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Österlund, Nicklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bianchi, Federica
    Ilag, Leopold
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Solvent-Assisted Paper Spray Ionization (SAPSI) for the Analysis of Biomolecules and BiofluidsManuskript (preprint) (Övrigt vetenskapligt)
  • 115.
    Riboni, Nicoló
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi. University of Parma, Italy.
    Quaranta, Alessandro
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Motwani, Hitesh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Österlund, Nickles
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bianchi, Federica
    Ilag, Leopold L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Solvent-Assisted Paper Spray Ionization Mass Spectrometry (SAPSI-MS) for the Analysis of Biomolecules and Biofluids2019Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 9, artikel-id 10296Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Paper Spray Ionization (PSI) is commonly applied for the analysis of small molecules, including drugs, metabolites, and pesticides in biological fluids, due to its high versatility, simplicity, and low costs. In this study, a new setup called Solvent Assisted Paper Spray Ionization (SAPSI), able to increase data acquisition time, signal stability, and repeatability, is proposed to overcome common PSI drawbacks. The setup relies on an integrated solution to provide ionization potential and constant solvent flow to the paper tip. Specifically, the ion source was connected to the instrument fluidics along with the voltage supply systems, ensuring a close control over the ionization conditions. SAPSI was successfully applied for the analysis of different classes of biomolecules: amyloidogenic peptides, proteins, and N-glycans. The prolonged analysis time allowed real-time monitoring of processes taking places on the paper tip, such as amyloid peptides aggregation and disaggregation phenomena. The enhanced signal stability allowed to discriminate protein species characterized by different post translational modifications and adducts with electrophilic compounds, both in aqueous solutions and in biofluids, such as serum and cerebrospinal fluid, without any sample pretreatment. In the next future, application to clinical relevant modifications, could lead to the development of quick and cost-effective diagnostic tools.

  • 116. Richman, Michal
    et al.
    Wilk, Sarah
    Chemerovski, Marina
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wahlström, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rahimipour, Shai
    In Vitro and Mechanistic Studies of an Antiamyloidogenic Self-Assembled Cyclic D,L-alpha-Peptide Architecture2013Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 135, nr 9, s. 3474-3484Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Misfolding of the A beta protein and its subsequent aggregation into toxic oligomers are related to Alzheimer's disease. Although peptides of various sequences can self-assemble into amyloid structures, these structures share common three-dimensional features that may promote their cross-reaction. Given the significant similarities between amyloids and the architecture of self-assembled cyclic D,L-alpha-peptide, we hypothesized that the latter may bind and stabilize a nontoxic form of A beta thereby preventing its aggregation into toxic forms. By screening a focused library of six-residue cyclic D,L-alpha-peptides and optimizing the activity of a lead peptide, we found one cyclic D,L-alpha-peptide (CP-2) that interacts strongly with A beta and inhibits its aggregation. In transmission electron microscopy, optimized thioflavin T and cell survival assays, CP-2 inhibits the formation of A beta aggregates, entirely disassembles preformed aggregated and fibrillar A beta, and protects rat pheochromocytoma PC12 cells from A beta toxicity, without inducing any toxicity by itself. Using various immunoassays, circular dichroism spectroscopy, photoinduced cross-linking of unmodified proteins (PICUP) combined with SDS/PAGE, and NMR, we probed the mechanisms underlying CP-2's antiamyloidogenic activity. NMR spectroscopy indicates that CP-2 interacts with A beta through its self-assembled conformation and induces weak secondary structure in A beta. Upon coincubation, CP-2 changes the aggregation pathway of A beta and alters its oligomer distribution by stabilizing small oligomers (1-3 mers). Our results support studies suggesting that toxic early oligomeric states of A beta may be composed of antiparallel beta-peptide structures and that the interaction of A beta with CP-2 promotes formation of more benign parallel beta-structures. Further studies will show whether these kinds of abiotic cyclic D,L-alpha-peptides are also beneficial as an intervention in related in vivo models.

  • 117. Roos, Elin
    et al.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. UCLA, USA.
    Meyer, Jeremy
    Sholts, Sabrina B.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. The National Institute of Chemical Physics and Biophysics, Estonia.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Roos, Per M.
    Amyotrophic Lateral Sclerosis After Exposure to Manganese from Traditional Medicine Procedures in Kenya2021Ingår i: Biological Trace Element Research, ISSN 0163-4984, E-ISSN 1559-0720, Vol. 199, s. 3618-3624Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss and widespread muscular atrophy. Despite intensive investigations on genetic and environmental factors, the cause of ALS remains unknown. Recent data suggest a role for metal exposures in ALS causation. In this study we present a patient who developed ALS after a traditional medical procedure in Kenya. The procedure involved insertion of a black metal powder into several subcutaneous cuts in the lower back. Four months later, general muscle weakness developed. Clinical and electrophysiological examinations detected widespread denervation consistent with ALS. The patient died from respiratory failure less than a year after the procedure. Scanning electron microscopy and X-ray diffraction analyses identified the black powder as potassium permanganate (KMnO4). A causative relationship between the systemic exposure to KMnO4 and ALS development can be suspected, especially as manganese is a well-known neurotoxicant previously found to be elevated in cerebrospinal fluid from ALS patients. Manganese neurotoxicity and exposure routes conveying this toxicity deserve further attention.

  • 118.
    Sahlin, Margareta
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Cho, Kyung-Bin
    Institutionen för biokemi och biofysik.
    Pötsch, Stephan
    Institutionen för biokemi och biofysik.
    Lytton, Simon D
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Huque, Yasmin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Gunther, Michael R
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Mason, Ronald P
    Gräslund, Astrid
    Institutionen för biokemi och biofysik.
    Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli.2002Ingår i: J Biol Inorg Chem, ISSN 0949-8257, Vol. 7, nr 1-2, s. 74-82Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.

  • 119. Shafaat, Hannah S.
    et al.
    Griese, Julia J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pantazis, Dimitrios A.
    Roos, Katarina
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum.
    Andersson, Charlotta S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Popovic-Bijelic, Ana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Siegbahn, Per E. M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum.
    Neese, Frank
    Lubitz, Wolfgang
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cox, Nicholas
    Electronic Structural Flexibility of Heterobimetallic Mn/Fe Cofactors: R2lox and R2c Proteins2014Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, nr 38, s. 13399-13409Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Hogbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn-III/Fe-III dimer linked by a mu-hydroxo/bis-mu-carboxylato bridging network. The Mn-III ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2lox(photo)) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn-III/Fe-III cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn-III ion, as best evidenced by a reorientation of its unique Mn-55 hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the mu-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform.

  • 120. Sigfridsson, Kajsa G. V.
    et al.
    Chernev, Petko
    Leidel, Nils
    Popovic-Bijelic, Ana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Haumann, Michael
    Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase2013Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 14, s. 9648-9661Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Prototypic dinuclear metal cofactors with varying metallation constitute a class of O-2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III) Fe(III) and Mn(III)-Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III, II) and (II, II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II, II) states are reached in < 1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II, II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II, II) cofactors, which deviate from the native structures functional in O-2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.

  • 121. Tanaka, Gen
    et al.
    Nakase, Ikuhiko
    Fukuda, Yasunori
    Masuda, Ryo
    Oishi, Shinya
    Shimura, Kazuya
    Kawaguchi, Yoshimasa
    Takatani-Nakase, Tomoka
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Okawa, Katsuya
    Matsuoka, Masao
    Fujii, Nobutaka
    Hatanaka, Yasumaru
    Futaki, Shiroh
    CXCR4 Stimulates Macropinocytosis: Implications for Cellular Uptake of Arginine-Rich Cell-Penetrating Peptides and HIV2012Ingår i: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 19, nr 11, s. 1437-1446Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis. R12 had activity to prevent viral infection for HIV-1(IIIB), a subtype of HIV-1 that uses CXCR4 as a coreceptor for entry into susceptible cells, whereas the addition of a macropinocytosis inhibitor, dimethylamiloride, resulted in enhancement of viral infection. The present study shows that CXCR4 triggers macropinocytosis, which may have implications for the cellular uptake of oligoarginine CPPs and internalization of HIV.

  • 122. Taqi, Malik Mumtaz
    et al.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Yamskova, Olga
    Madani, Fatemeh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bazov, Igor
    Luo, Jinghui
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Zubarev, Roman
    Verbeek, Dineke
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bakalkin, Georgy
    Conformation Effects of CpG Methylation on Single-Stranded DNA Oligonucleotides: Analysis of the Opioid Peptide Dynorphin-Coding Sequences2012Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 7, nr 6, s. e39605-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Single-stranded DNA (ssDNA) is characterized by high conformational flexibility that allows these molecules to adopt a variety of conformations. Here we used native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy to show that cytosine methylation at CpG sites affects the conformational flexibility of short ssDNA molecules. The CpG containing 37-nucleotide PDYN (prodynorphin) fragments were used as model molecules. The presence of secondary DNA structures was evident from differences in oligonucleotide mobilities on PAGE, from CD spectra, and from formation of A-T, G-C, and non-canonical G-T base pairs observed by NMR spectroscopy. The oligonucleotides displayed secondary structures at 4 degrees C, and some also at 37 degrees C. Methylation at CpG sites prompted sequence-dependent formation of novel conformations, or shifted the equilibrium between different existing ssDNA conformations. The effects of methylation on gel mobility and base pairing were comparable in strength to the effects induced by point mutations in the DNA sequences. The conformational effects of methylation may be relevant for epigenetic regulatory events in a chromatin context, including DNA-protein or DNA-DNA recognition in the course of gene transcription, and DNA replication and recombination when double-stranded DNA is unwinded to ssDNA.

  • 123.
    Teixeira, Pedro F.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Masuyer, Geoffrey
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pinho, Catarina M.
    Branca, Rui M. M.
    Kmiec, Beata
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wallin, Cecilia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berntsson, Ronnie P. -A.
    Ankarcrona, Maria
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lehtiö, Janne
    Stenmark, Pål
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin2018Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, nr 3, s. 348-362Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Proteolysis plays an important role in mitochondria! biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLN(E475Q) in complex with the products of neurotensin cleavage at 2.7 angstrom revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-beta peptide, A beta 1-40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment A beta 35-40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.

  • 124.
    Tiiman, Ann
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. The National Institute of Chemical Physics and Biophysics, Estonia.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vukojevic, Vladana
    Heterogeneity and Turnover of Intermediates during Amyloid-beta (A beta) Peptide Aggregation Studied by Fluorescence Correlation Spectroscopy2015Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, nr 49, s. 7203-7211Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Self-assembly of amyloid beta (A beta) peptide molecules into large aggregates is a naturally occurring process driven in aqueous solution by a dynamic interplay between hydrophobic interactions among A beta molecules, which promote aggregation, and steric and overall electrostatic hindrance, which stifles it. A beta self-association is entropically unfavorable, as it implies order increase in the system, but under favorable kinetic conditions, the process proceeds at appreciable rates, yielding A beta aggregates of different sizes and structures. Despite the great relevance and extensive research efforts, detailed kinetic mechanisms underlying A beta aggregation remain only partially understood. In this study, fluorescence correlation spectroscopy (FCS) and Thioflavin T (ThT) were used to monitor the time dependent growth of structured aggregates and characterize multiple components during the aggregation of A beta peptides in a heterogeneous aqueous solution. To this aim, we collected data during a relatively large number of observation periods, 30 consecutive measurements lasting 10 s each, at what we consider to be a constant time point in the slow aggregation process. This approach enabled monitoring the formation of nanomolar concentrations of structured amyloid aggregates and demonstrated the changing distribution of amyloid aggregate sizes throughout the aggregation process. We identified aggregates of different sizes with molecular weight from 260 to more than 1 x 10(6) kDa and revealed the hitherto unobserved kinetic turnover of intermediates during A beta aggregation. The effect of different A beta concentrations, A beta:ThT ratios, differences between the 40 (A beta 40) and 42 (A beta 42) residue long variants of A beta, and the effect of stirring were also examined.

  • 125. Tiiman, Ann
    et al.
    Jelic, Vesna
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. The National Institute of Chemical Physics and Biophysics, Estonia..
    Jaremo, Petter
    Bogdanovic, Nenad
    Rigler, Rudolf
    Terenius, Lars
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vukojevic, Vladana
    Amyloidogenic Nanoplaques in Blood Serum of Patients with Alzheimer's Disease Revealed by Time-Resolved Thioflavin T Fluorescence Intensity Fluctuation Analysis2019Ingår i: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 68, nr 2, s. 571-582Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Biomarkers are central to current research on molecular mechanisms underlying Alzheimer's disease (AD). Their further development is of paramount importance for understanding pathophysiological processes that eventually lead to disease onset. Biomarkers are also crucial for early disease detection, before clinical manifestation, and for development of new disease modifying therapies. Objective: The overall aim of this work is to develop a minimally invasive method for fast, ultra-sensitive and cost-effective detection of structurally modified peptide/protein self-assemblies in the peripheral blood and in other biological fluids. Specifically, we focus here on using this method to detect structured amyloidogenic oligomeric aggregates in the blood serum of apparently healthy individuals and patients in early AD stage, and measure their concentration and size. Methods: Time-resolved detection of Thioflavin T (ThT) fluorescence intensity fluctuations in a sub-femtoliter observation volume element was used to identify in blood serum ThT-active structured amyloidogenic oligomeric aggregates, hereafter called nanoplaques, and measure with single-particle sensitivity their concentration and size. Results: The concentration and size of structured amyloidogenic nanoplaques are significantly higher in the blood serum of individuals diagnosed with AD than in control subjects. Conclusion: A new method with the ultimate, single-particle sensitivity was successfully developed. The proposed approach neither relies on the use of immune-based probes, nor on the use of radiotracers, signal-amplification or protein separation techniques, and provides a minimally invasive test for fast and cost-effective early determination of structurally modified peptides/proteins in the peripheral blood, as shown here, but also in other biological fluids.

  • 126.
    Tiiman, Ann
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Luo, Jinghui
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Oxford, UK.
    Wallin, Cecilia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Olsson, Lisa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindgren, Joel
    Jarvet, Jϋri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. The National Institute of Chemical Physics and Biophysics, Estonia.
    Roos, Per
    Sholts, Sabrina B.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. National Museum of Natural History, USA.
    Rahimipour, Shai
    Abrahams, Jan Pieter
    Eriksson Karlström, Amelie
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Specific Binding of Cu(II) Ions to Amyloid-Beta Peptides Bound to Aggregation-Inhibiting Molecules or SDS Micelles Creates Complexes that Generate Radical Oxygen Species2016Ingår i: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 54, nr 3, s. 971-982Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aggregation of the amyloid-beta (A beta) peptide into insoluble plaques is a major factor in Alzheimer's disease (AD) pathology. Another major factor in AD is arguably metal ions, as metal dyshomeostasis is observed in AD patients, metal ions modulate A beta aggregation, and AD plaques contain numerous metals including redox-active Cu and Fe ions. In vivo, A beta is found in various cellular locations including membranes. So far, Cu(II)/A beta interactions and ROS generation have not been investigated in a membrane environment. Here, we study Cu(II) and Zn(II) interactions with A beta bound to SDS micelles or to engineered aggregation-inhibiting molecules (the cyclic peptide CP-2 and the Z(A beta 3)(12-58) Y18L Affibody molecule). In all studied systems the A beta N-terminal segment was found to be unbound, unstructured, and free to bind metal ions. In SDS micelles, A beta was found to bind Cu(II) and Zn(II) with the same ligands and the same K-D as in aqueous solution. ROS was generated in all Cu(II)/A beta complexes. These results indicate that binding of A beta to membranes, drugs, and other entities that do not interact with the A beta N-terminal part, appears not to compromise the N-terminal segment's ability to bind metal ions, nor impede the capacity of N-terminally bound Cu(II) to generate ROS.

  • 127.
    Tiklová, Katarína
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Senti, Kirsten-André
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Wang, Shenqiu
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Samakovlis, Christos
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila2010Ingår i: Nature Cell Biology, ISSN 1465-7392, E-ISSN 1476-4679, Vol. 12, nr 11, s. 1071-1077Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Iron is an essential element in many biological processes. In vertebrates, serum transferrin is the major supplier of iron to tissues, but the function of additional transferrin-like proteins remains poorly understood. Melanotransferrin (MTf) is a phylogenetically conserved, iron-binding epithelial protein. Elevated MTf levels have been implicated in melanoma pathogenesis. Here, we present a functional analysis of MTf in Drosophila melanogaster. Similarly to its human homologue, Drosophila MTf is a lipid-modified, iron-binding protein attached to epithelial cell membranes, and is a component of the septate junctions that form the paracellular permeability barrier in epithelial tissues. We demonstrate that septate junction assembly during epithelial maturation relies on endocytosis and apicolateral recycling of iron-bound MTf. Mouse MTf complements the defects of Drosophila MTf mutants. Drosophila provides the first genetic model for the functional dissection of MTf in epithelial junction assembly and morphogenesis.

  • 128.
    Torrents, Eduard
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Sahlin, Margareta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Biglino, Daniele
    Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Institutionen för biokemi och biofysik.
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Efficient growth inhibition of Bacillus anthracis by knocking out the ribonucleotide reductase tyrosyl radical.2005Ingår i: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 102, nr 50, s. 17946-51Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    Bacillus anthracis, the causative agent of anthrax, is a worldwide problem because of the need for effective treatment of respiratory infections shortly after exposure. One potential key enzyme of B. anthracis to be targeted by antiproliferative drugs is ribonucleotide reductase. It provides deoxyribonucleotides for DNA synthesis needed for spore germination and growth of the pathogen. We have cloned, purified, and characterized the tyrosyl radical-carrying NrdF component of B. anthracis class Ib ribonucleotide reductase. Its EPR spectrum points to a hitherto unknown three-dimensional geometry of the radical side chain with a 60 degrees rotational angle of C(alpha)-(C(beta)-C(1))-plane of the aromatic ring. The unusual relaxation behavior of the radical signal and its apparent lack of line broadening at room temperature suggest a weak interaction with the nearby diiron site and the presence of a water molecule plausibly bridging the phenolic oxygen of the radical to a ligand of the diiron site. We show that B. anthracis cells are surprisingly resistant to the radical scavenger hydroxyurea in current use as an antiproliferative drug, even though its NrdF radical is efficiently scavenged in vitro. Importantly, the antioxidants hydroxylamine and N-methyl hydroxylamine scavenge the radical several orders of magnitude faster and prevent B. anthracis growth at several hundred-fold lower concentrations compared with hydroxyurea. Phylogenetically, the B. anthracis NrdF protein clusters together with NrdFs from the pathogens Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, and Staphylococcus epidermidis. We suggest the potential use of N-hydroxylamines in combination therapies against infections by B. anthracis and closely related pathogens.

  • 129.
    Unnerståle, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Madani, Fatemeh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane-perturbing properties of two Arg-rich paddle domains from voltage-gated sensors in the KvAP and HsapBK K+ channels2012Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, nr 19, s. 3982-3992Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Voltage-gated K+ channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a “paddle” domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains, KvAPp from the Kv channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy. We show that both paddle domains induce calcein leakage in large unilamellar vesicles, and we suggest that this leakage represents a general thinning of the bilayer, making movement of the whole paddle domain plausible. The fact that HsapBKp induces more leakage than KvAPp may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic–hydrophobic interface and disturb tight packing. In magnetically aligned bicelles, KvAPp increases the level of order along the whole acyl chain, while HsapBKp affects the morphology, also indicating that KvAPp adapts more to the lipid environment. Nuclear magnetic resonance (NMR) relaxation measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is well-structured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility by which HsapBKp can undergo structural transitions is also shown by relaxation NMR, which may be important for the gating mechanism.

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  • 130.
    Vasconcelos, Luís
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Madani, Fatemeh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Arukuusk, Piret
    Pärnaste, Ly
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Effects of cargo molecules on membrane perturbation caused by transportan10 based cell-penetrating peptides2014Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1838, nr 12, s. 3118-3129Artikel i tidskrift (Refereegranskat)
    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.

  • 131.
    Voevodskaya, Nina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lendzian, Friedhelm
    Ehrenberg, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    High catalytic activity achieved with a mixed manganese-iron site in protein R2 of Chlamydia ribonucleotide reductase2007Konferensbidrag (Övrig (populärvetenskap, debatt, mm))
  • 132. Vukojevic, Vladana
    et al.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bakalkin, Georgy
    Fluorescence Imaging with Single-Molecule Sensitivity and Fluorescence Correlation Spectroscopy of Cell-Penetrating Neuropeptides2011Ingår i: Neuropeptides: methods and protocols / [ed] Merighi, A., Humana Press, 2011, s. 147-170Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Neuropeptide plasma membrane interactions in the absence of a corresponding specific receptor may result in neuropeptide translocation into the cell. Trans location across the plasma membrane may represent a previously unknown mechanism by which neuropeptides can signal information to the cell interior. We introduce here two complementary optical methods with single-molecule sensitivity, fluorescence imaging with avalanche photodiode detectors (APD imaging) and fluorescence correlation spectroscopy (FCS), and demonstrate how they may be applied for the analysis of neuropeptide ability to penetrate into live cells in real time. APD imaging enables us to visualize fluorescently labeled neuropeptide molecules at very low, physiologically relevant concentrations, whereas FCS enables us to characterize quantitatively their concentration and diffusion properties in different cellular compartments. Application of these methodologies for the analysis of the endogenous opioid peptide dynorphin A (Dyn A), a ligand for the kappa-opioid receptor (KOP), demonstrated that this neuropeptide may translocate across the plasma membrane of living cells and enter the cellular interior without binding to its cognate receptor.

  • 133.
    Wahlström, Anna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cukalevski, Risto
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, Jueri
    Onagi, Hideki
    Rebek, Julius, Jr.
    Linse, Sara
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Specific binding of a beta cyclodextrin dimer to the amyloid beta peptide modulates the peptide aggregation process2012Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, nr 21, s. 4280-4289Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alzheimer's disease involves progressive neuronal loss. Linked to the disease is the amyloid beta (A beta) peptide, a 38-43-amino acid peptide found in extracellular amyloid plaques in the brain. Cyclodextrins are nontoxic, cone-shaped oligosaccharides with a hydrophilic exterior and a hydrophobic cavity making them suitable hosts for aromatic guest molecules in water. beta-Cyclodextrin consists of seven alpha-D-glucopyranoside units and has been shown to reduce the level of fibrillation and neurotoxicity of A beta. We have studied the interaction between A beta and a beta-cyclodextrin dimer, consisting of two beta-cyclodextrin monomers connected by a flexible linker. The beta-cyclodextrin monomer has been found to interact with A beta(1-40) at sites Y10, F19, and/or F20 with a dissociation constant (K-D) of 3.9 +/- 2.0 mM. Here H-1-N-15 and H-1-C-13 heteronuclear single-quantum correlation nuclear magnetic resonance (NMR) spectra show that in addition, the beta-cyclodextrin monomer and dimer bind to the histidines. NMR translational diffusion experiments reveal the increased affinity of the beta-cyclodextrin dimer (apparent K-D of 1.1 +/- .5 mM) for A beta(1-40) compared to that of the beta-cyclodextrin monomer. Kinetic aggregation experiments based on thioflavin T fluorescence indicate that the dimer at 0.05-5 mM decreases the lag time of A beta aggregation, while a concentration of 10 mM increases the lag time. The beta-cyclodextrin monomer at a high concentration decreases the lag time of the aggregation. We conclude that cyclodextrin monomers and dimers have specific, modulating effects on the A beta(1-40) aggregation process. Transmission electron microscopy shows that the regular fibrillar aggregates formed by A beta(1-40) alone are replaced by a major fraction of amorphous aggregates in the presence of the beta-cyclodextrin dimer.

  • 134.
    Wahlström, Anna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cukalevski, Risto
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, Jüri
    Onagi, Hideki
    Rebek Jr., Julius
    Linse, Sara
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Specific binding of an engineered β-cyclodextrin dimer to the amyloid β peptide modulates the peptide aggregation processManuskript (preprint) (Övrigt vetenskapligt)
  • 135.
    Wahlström, Anna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hugonin, Loïc
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Perálvarez-Marín, Alex
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, Jüri
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Secondary structure conversions of Alzheimer’s Aβ(1–40) peptide induced by membrane-mimicking detergents2008Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, nr 20, s. 5117-5128Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The amyloid β peptide (Aβ) with 39–42 residues is the major component of amyloid plaques found in brains of Alzheimer’s disease patients, and soluble oligomeric peptide aggregates mediate toxic effects on neurons. The Aβ aggregation involves a conformational change of the peptide structure to β-sheet. In the present study, we report on the effect of detergents on the structure transitions of Aβ, to mimic the effects that biomembranes may have. In vitro, monomeric Aβ(1–40) in a dilute aqueous solution is weakly structured. By gradually adding small amounts of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate to a dilute aqueous solution, Aβ(1–40) is converted to β-sheet, as observed by CD at 3 °C and 20 °C. The transition is mainly a two-state process, as revealed by approximately isodichroic points in the titrations. Aβ(1–40) loses almost all NMR signals at dodecyl sulfate concentrations giving rise to the optimal β-sheet content (approximate detergent/peptide ratio = 20). Under these conditions, thioflavin T fluorescence measurements indicate a maximum of aggregated amyloid-like structures. The loss of NMR signals suggests that these are also involved in intermediate chemical exchange. Transverse relaxation optimized spectroscopy NMR spectra indicate that the C-terminal residues are more dynamic than the others. By further addition of SDS or lithium dodecyl sulfate reaching concentrations close to the critical micellar concentration, CD, NMR and FTIR spectra show that the peptide rearranges to form a micelle-bound structure with α-helical segments, similar to the secondary structures formed when a high concentration of detergent is added directly to the peptide solution.

  • 136.
    Wallin, Cecilia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Friedemann, Merlin
    Sholts, Sabrina B.
    Noormägi, Andra
    Svantesson, Teodor
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. The National Institute of Chemical Physics and Biophysics, Estonia.
    Roos, Per M.
    Palumaa, Peep
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mercury and Alzheimer's Disease: Hg(II) Ions Display Specific Binding to the Amyloid-β Peptide and Hinder Its Fibrillization2020Ingår i: Biomolecules, E-ISSN 2218-273X, Vol. 10, nr 1, artikel-id 44Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Brains and blood of Alzheimer's disease (AD) patients have shown elevated mercury concentrations, but potential involvement of mercury exposure in AD pathogenesis has not been studied at the molecular level. The pathological hallmark of AD brains is deposition of amyloid plaques, consisting mainly of amyloid-beta (A beta) peptides aggregated into amyloid fibrils. A beta peptide fibrillization is known to be modulated by metal ions such as Cu(II) and Zn(II). Here, we study in vitro the interactions between A beta peptides and Hg(II) ions by multiple biophysical techniques. Fluorescence spectroscopy and atomic force microscopy (AFM) show that Hg(II) ions have a concentration-dependent inhibiting effect on A beta fibrillization: at a 1:1 A betaHg(II) ratio only non-fibrillar A beta aggregates are formed. NMR spectroscopy shows that Hg(II) ions interact with the N-terminal region of A beta(1-40) with a micromolar affinity, likely via a binding mode similar to that for Cu(II) and Zn(II) ions, i.e., mainly via the histidine residues His6, His13, and His14. Thus, together with Cu(II), Fe(II), Mn(II), Pb(IV), and Zn(II) ions, Hg(II) belongs to a family of metal ions that display residue-specific binding interactions with A beta peptides and modulate their aggregation processes.

  • 137.
    Wallin, Cecilia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hiruma, Yoshitaka
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Huvent, Isabelle
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abrahams, Jan Pieter
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lippens, Guy
    Luo, Jinghui
    The Neuronal Tau Protein Blocks in Vitro Fibrillation of the Amyloid-beta (A beta) Peptide at the Oligomeric Stage2018Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, nr 26, s. 8138-8146Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In Alzheimer's disease, amyloid-beta (A beta) plaques and tau neurofibrillary tangles are the two pathological hallmarks. The co-occurrence and combined reciprocal pathological effects of A beta and tau protein aggregation have been observed in animal models of the disease. However, the molecular mechanism of their interaction remain unknown. Using a variety of biophysical measurements, we here show that the native full-length tau protein solubilizes the A beta(40) peptide and prevents its fibrillation. The tau protein delays the amyloid fibrillation of the A beta(40) peptide at substoichiometric ratios, showing different binding affinities toward the different stages of the aggregated A beta(40) peptides. The A beta monomer structure remains random coil in the presence of tau, as observed by nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy and photoinduced cross-linking methods. We propose a potential interaction mechanism for the influence of tau on A beta fibrillation.

  • 138.
    Wallin, Cecilia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biverstål, Henrik
    Wärmländer, Sebastian
    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.
    Abelein, Axel
    Metal ion coordination delays amyloid-β peptide self-assembly by forming an aggregation-inert complex2020Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 295, nr 21, s. 7224-7234Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A detailed understanding of the molecular pathways for amyloid-β (Aβ) peptide aggregation from monomers into amyloid fibrils, a hallmark of Alzheimer’s disease, is crucial for the development of diagnostic and therapeutic strategies. We investigate the molecular details of peptide fibrillization in vitro by perturbing this process through addition of differently charged metal ions. Here, we used a monovalent probe, the silver ion, that, similarly to divalent metal ions, binds to monomeric Aβ peptide and efficiently modulates Aβ fibrillization. On the basis of our findings, combined with our previous results on divalent zinc ions, we propose a model that links the microscopic metal ion binding to Aβ monomers to its macroscopic impact on the peptide self-assembly observed in bulk experiments. We found that sub-stoichiometric concentrations of the investigated metal ions bind specifically to the N-terminal region of Aβ, forming a dynamic, partially compact complex. The metal ion bound state appears to be incapable of aggregation, effectively reducing the available monomeric Aβ pool for incorporation into fibrils. This is especially reflected in a decreased fibril-end elongation rate. However, since the bound state is significantly less stable than the amyloid state, Aβ peptides are only transiently redirected from fibril formation and eventually almost all Aβ monomers are integrated into fibrils. Taken together, these findings unravel the mechanistic consequences of delaying Aβ aggregation via weak metal ion binding, quantitatively linking the contributions of specific interactions of metal ions with monomeric Aβ to their effects on bulk aggregation.

  • 139.
    Wallin, Cecilia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kulkarni, Yashraj S.
    Abelein, Axel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Karolinska Institutet, Sweden.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. The National Institute of Chemical Physics and Biophysics, Estonia.
    Liao, Qinghua
    Strodel, Birgit
    Olsson, Lisa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Luo, Jinghui
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Oxford, UK.
    Abrahams, Jan Pieter
    Sholts, Sabrina B.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. National Museum of Natural History, USA.
    Roos, Per M.
    Kamerlin, Shina C. L.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Characterization of Mn(II) ion binding to the amyloid-beta peptide in Alzheimer's disease2016Ingår i: Journal of Trace Elements in Medicine and Biology, ISSN 0946-672X, E-ISSN 1878-3252, Vol. 38, s. 183-193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Growing evidence links neurodegenerative diseases to metal exposure. Aberrant metal ion concentrations have been noted in Alzheimer's disease (AD) brains, yet the role of metals in AD pathogenesis remains unresolved. A major factor in AD pathogenesis is considered to be aggregation of and amyloid formation by amyloid-beta (A beta) peptides. Previous studies have shown that A beta displays specific binding to Cu(II) and Zn(II) ions, and such binding has been shown to modulate A beta aggregation. Here, we use nuclear magnetic resonance (NMR) spectroscopy to show that Mn(II) ions also bind to the N-terminal part of the A beta(1-40) peptide, with a weak binding affinity in the milli- to micromolar range. Circular dichroism (CD) spectroscopy, solid state atomic force microscopy (AFM), fluorescence spectroscopy, and molecular modeling suggest that the weak binding of Mn(II) to A beta may not have a large effect on the peptide's aggregation into amyloid fibrils. However, identification of an additional metal ion displaying A beta binding reveals more complex AD metal chemistry than has been previously considered in the literature.

  • 140.
    Wallin, Cecilia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Luo, Jinghui
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Oxford, UK.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. The National Institute of Chemical Physics and Biophysics, Estonia.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The Amyloid-beta Peptide in Amyloid Formation Processes: Interactions with Blood Proteins and Naturally Occurring Metal Ions2017Ingår i: Israel Journal of Chemistry, ISSN 0021-2148, Vol. 57, nr 7-8, s. 674-685Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    This review describes interactions between the amyloid- peptide (A) involved in Alzheimer's disease (AD) and endogenous metal ions and proteins, with an emphasis on future potential drug therapies and targets. AD is characterised by loss of neurons, memory, and cognitive functions, and by formation of cerebral senile plaque deposits. These plaques consist mainly of aggregated A peptides. AD pathology includes a) on the molecular level imbalanced concentrations of A peptides and metal ions, and formation of amyloid structures, and b) on the physiological level a combination of inflammatory responses and oxidative stress effects causing neuronal death. Interestingly, certain blood proteins and metal ions can affect the A amyloid aggregation process. These interactions are the topics of the present review. A deeper understanding of these interactions could facilitate new therapeutic strategies against AD. Previous therapeutic approaches and trials are also briefly described.

  • 141.
    Wallin, Cecilia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sholts, Sabrina B.
    Österlund, Nicklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Luo, Jinghui
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. The National Institute of Chemical Physics and Biophysics, Estonia.
    Roos, Per M.
    Ilag, Leopold
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Alzheimer's disease and cigarette smoke components: effects of nicotine, PAHs, and Cd(II), Cr(III), Pb(II), Pb(IV) ions on amyloid-beta peptide aggregation2017Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 7, artikel-id 14423Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cigarette smoking is a significant risk factor for Alzheimer’s disease (AD), which is associated with extracellular brain deposits of amyloid plaques containing aggregated amyloid-β (Aβ) peptides. Aβ aggregation occurs via multiple pathways that can be influenced by various compounds. Here, we used AFM imaging and NMR, fluorescence, and mass spectrometry to monitor in vitro how Aβ aggregation is affected by the cigarette-related compounds nicotine, polycyclic aromatic hydrocarbons (PAHs) with one to five aromatic rings, and the metal ions Cd(II), Cr(III), Pb(II), and Pb(IV). All PAHs and metal ions modulated the Aβ aggregation process. Cd(II), Cr(III), and Pb(II) ions displayed general electrostatic interactions with Aβ, whereas Pb(IV) ions showed specific transient binding coordination to the N-terminal Aβ segment. Thus, Pb(IV) ions are especially prone to interact with Aβ and affect its aggregation. While Pb(IV) ions affected mainly Aβ dimer and trimer formation, hydrophobic toluene mainly affected formation of larger aggregates such as tetramers. The uncharged and hydrophilic nicotine molecule showed no direct interactions with Aβ, nor did it affect Aβ aggregation. Our Aβ interaction results suggest a molecular rationale for the higher AD prevalence among smokers, and indicate that certain forms of lead in particular may constitute an environmental risk factor for AD.

  • 142. Wang, Chao
    et al.