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  • 51.
    Gräslund, Astrid
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ehrenberg, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Tyrosyl radical, diiron center and enzyme mechanism in ribonucleotide reductase2007Ingår i: Appl. Magn. Reson., nr 31, s. 447-455Artikel i tidskrift (Refereegranskat)
  • 52.
    Gräslund, Astrid
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Testing membrane interactions of CPPs2011Ingår i: Methods in molecular biology (Clifton, N.J.), ISSN 1940-6029, Vol. 683, s. 33-40Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The chapter deals with some biophysical methods used for investigating CPP-induced changes in membrane properties by spectroscopy methods such as fluorescence or NMR and methods used for probing CPP-induced leakage in membranes. Some useful model systems for biomembranes are described. These include large unilamellar phospholipid vesicles (LUVs) of well-defined size (diameter typically 100 nm). A protocol for the preparation of such vesicles is included. The leakage studies make use of LUVs with entrapped dye molecules. The NMR studies make use of mixed micelles (bicelles) as a membrane mimetic system, which can be oriented in the magnetic field of the spectrometer.

  • 53.
    Guterstam, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Madani, Fatemeh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hirose, Hisaaki
    Takeuchi, Toshihide
    Futaki, Shiroh
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate2009Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, nr 12, s. 2509-2517Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with various hydrophobicity and arginine content is investigated. The cellular CPP-uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2’-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.

  • 54.
    Hammerstad, Marta
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Oslo, Norway.
    Rohr, Asmund K.
    Andersen, Niels H.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Andersson, K. Kristoffer
    The class Ib ribonucleotide reductase from Mycobacterium tuberculosis has two active R2F subunits2014Ingår i: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, nr 6, s. 893-902Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class Ib RNRs require either a diiron-tyrosyl radical (Y center dot) or a dimanganese-Y center dot cofactor in their R2F subunit to initiate ribonucleotide reduction in the R1 subunit. Mycobacterium tuberculosis, the causative agent of tuberculosis, contains two genes, nrdF1 and nrdF2, encoding the small subunits R2F-1 and R2F-2, respectively, where the latter has been thought to serve as the only active small subunit in the M. tuberculosis class Ib RNR. Here, we present evidence for the presence of an active Fe (2) (III) -Y center dot cofactor in the M. tuberculosis RNR R2F-1 small subunit, supported and characterized by UV-vis, X-band electron paramagnetic resonance, and resonance Raman spectroscopy, showing features similar to those for the M. tuberculosis R2F-2-Fe (2) (III) -Y center dot cofactor. We also report enzymatic activity of Fe (2) (III) -R2F-1 when assayed with R1, and suggest that the active M. tuberculosis class Ib RNR can use two different small subunits, R2F-1 and R2F-2, with similar activity.

  • 55. Heffeter, P.
    et al.
    Popovic-Bijelic, Ana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Saiko, P.
    Dornetshuber, R.
    Jungwirth, U.
    Voevodskaya, Nina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biglino, D.
    Jakupec, M. A.
    Elbling, L.
    Micksche, M.
    Szekeres, T.
    Keppler, B. K.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berger, W.
    Ribonucleotide Reductase as One Important Target of [Tris(1,10- phenanthroline)lanthanum(III)] Trithiocyanate (KP772)2009Ingår i: Current Cancer Drug Targets, ISSN 1568-0096, E-ISSN 1873-5576, Vol. 9, nr 5, s. 595-607Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    KP772 is a new lanthanum complex containing three 1,10-phenathroline molecules. Recently, we have demonstrated that the promising in vitro and in vivo anticancer properties of KP772 are based on p53-independent G(0)G(1) arrest and apoptosis induction. A National Cancer Institute (NCI) screen revealed significant correlation of KP772 activity with that of the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU). Consequently, this study aimed to investigate whether KP772 targets DNA synthesis in tumor cells by RR inhibition. Indeed, KP772 treatment led to significant reduction of cytidine incorporation paralleled by a decrease of deoxynucleoside triphosphate (dNTP) pools. This strongly indicates disruption of RR activity. Moreover, KP772 protected against oxidative stress, suggesting that this drug might interfere with RR by interaction with the tyrosyl radical in subunit R2. Additionally, several observations (e.g. increase of transferrin receptor expression and protective effect of iron preloading) indicate that KP772 interferes with cellular iron homeostasis. Accordingly, co-incubation of Fe(II) with KP772 led to generation of a coloured iron complex (Fe-KP772) in cell free systems. In electron paramagnetic resonance (EPR) measurements of mouse R2 subunits, KP772 disrupted the tyrosyl radical while Fe-KP772 had no significant effects. Moreover, coincubation of KP772 with iron-loaded R2 led to formation of Fe-KP772 suggesting chelation of RR-bound Fe(II). Summarizing, our data prove that KP772 inhibits RR by targeting the iron centre of the R2 subunit. As also Fe-KP772 as well as free lanthanum exert significant -though less pronounced- cytotoxic/static activities, additional mechanisms are likely to synergise with RR inhibition in the promising anticancer activity of KP772.

  • 56. Henning-Knechtel, Anja
    et al.
    Kumar, Sunil
    Wallin, Cecilia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Król, Sylwia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    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. The National Institute of Chemical Physics and Biophysics, Estonia.
    Esposito, Gennaro
    Kirmizialtin, Serdal
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hamilton, Andrew D.
    Magzoub, Mazin
    Designed Cell-Penetrating Peptide Inhibitors of Amyloid-beta Aggregation and Cytotoxicity2020Ingår i: Cell Reports Physical Science, E-ISSN 2666-3864, Vol. 1, nr 2, artikel-id 100014Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Amyloid proteins and peptides are a major contributing factor to the development of various neurodegenerative disorders, including Alzheimer’s and prion diseases. Previously, a designed cell-penetrating peptide (CPP) comprising a hydrophobic signal sequence followed by a prion protein (PrP)-derived polycationic sequence (PrP23–28: KKRPKP) was shown to have potent anti-prion properties. Here, we extend this approach toward the amyloid-beta (Aβ) peptide amyloid formation, which is associated with Alzheimer’s disease. We characterized the interactions of the CPP with Aβ using complementary in vitro and in silico experiments. We report that the CPP stabilizes Aβ in a non-amyloid state and inhibits Aβ-induced neurotoxicity. Moreover, replacing PrP23–28 with a corresponding segment from Aβ results in a construct with similar CPP functionality and antagonism of Aβ aggregation and neurotoxicity. Our findings reveal a general underlying principle for inhibition of pathogenic protein aggregation that may facilitate the design of CPP-based therapeutics for amyloid diseases.

  • 57. Horvath, Istvan
    et al.
    Iashchishyn, Igor A.
    Moskalenko, Roman A.
    Wang, Chao
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wallin, Cecilia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kovacs, Gabor G.
    Morozova-Roche, Ludmilla A.
    Co-aggregation of pro-inflammatory S100A9 with alpha-synuclein in Parkinson's disease: ex vivo and in vitro studies2018Ingår i: Journal of Neuroinflammation, ISSN 1742-2094, E-ISSN 1742-2094, Vol. 15, artikel-id 172Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Chronic neuroinflammation is a hallmark of Parkinson's disease (PD) pathophysiology, associated with increased levels of pro-inflammatory factors in PD brain tissues. The pro-inflammatory mediator and highly amyloidogenic protein S100A9 is involved in the amyloid-neuroinflammatory cascade in Alzheimer's disease. This is the first report on the co-aggregation of alpha-synuclein (alpha-syn) and S100A9 both in vitro and ex vivo in PD brain. Methods: Single and sequential immunohistochemistry, immunofluorescence, scanning electron and atomic force (AFM) microscopies were used to analyze the ex vivo PD brain tissues for S100A9 and alpha-syn location and aggregation. In vitro studies revealing S100A9 and alpha-syn interaction and co-aggregation were conducted by NMR, circular dichroism, Thioflavin-T fluorescence, AFM, and surface plasmon resonance methods. Results: Co-localized and co-aggregated S100A9 and alpha-syn were found in 20% Lewy bodies and 77% neuronal cells in the substantia nigra; both proteins were also observed in Lewy bodies in PD frontal lobe (Braak stages 4-6). Lewy bodies were characterized by ca. 10-23 mu m outer diameter, with S100A9 and alpha-syn being co-localized in the same lamellar structures. S100A9 was also detected in neurons and blood vessels of the aged patients without PD, but in much lesser extent. In vitro S100A9 and alpha-syn were shown to interact with each other via the alpha-syn C-terminus with an apparent dissociation constant of ca. 5 mu M. Their co-aggregation occurred significantly faster and led to formation of larger amyloid aggregates than the self-assembly of individual proteins. S100A9 amyloid oligomers were more toxic than those of alpha-syn, while co-aggregation of both proteins mitigated the cytotoxicity of S100A9 oligomers. Conclusions: We suggest that sustained neuroinflammation promoting the spread of amyloidogenic S100A9 in the brain tissues may trigger the amyloid cascade involving alpha-syn and S100A9 and leading to PD, similar to the effect of S100A9 and A beta co-aggregation in Alzheimer's disease. The finding of S100A9 involvement in PD may open a new avenue for therapeutic interventions targeting S100A9 and preventing its amyloid self-assembly in affected brain tissues.

  • 58.
    Hugonin, Loïc
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Barth, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Perálvarez-Marín, Alex
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Secondary structure transitions and aggregation induced in dynorphin neuropeptides by the detergent sodium dodecyl sulfate.2008Ingår i: Biochim Biophys Acta, ISSN 0006-3002, Vol. 1778, nr 11, s. 2580-7Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Dynorphins, endogeneous opioid neuropeptides, function as ligands to the opioid kappa receptors and also induce non-opioid effects in neurons, probably related to direct membrane interactions. We have characterized the structure transitions of dynorphins (big dynorphin, dynorphin A and dynorphin B) induced by the detergent sodium dodecyl sulfate (SDS). In SDS titrations monitored by circular dichroism, we observed secondary structure conversions of the peptides from random coil to alpha-helix with a highly aggregated intermediate. As determined by Fourier transform infrared spectroscopy, this intermediate exhibited beta-sheet structure for dynorphin B and big dynorphin. In contrast, aggregated dynorphin A was alpha-helical without considerable beta-sheet content. Hydrophobicity analysis indicates that the YGGFLRR motif present in all dynorphins is prone to be inserted in the membrane. Comparing big dynorphin with dynorphin A and dynorphin B, we suggest that the potent neurotoxicity of big dynorphin could be related to the combination of amino acid sequences and secondary structure propensities of dynorphin A and dynorphin B, which may generate a synergistic effect for big dynorphin membrane perturbing properties. The induced aggregated alpha-helix of dynorphin A is also correlated with membrane perturbations, whereas the beta-sheet of dynorphin B does not correlate with membrane perturbations.

  • 59. Khaled, Mohammed
    et al.
    Rönnbäck, Isabel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ilag, Leopold L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Strodel, Birgit
    Österlund, Nicklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK). Karolinska Institutet, Sweden.
    A Hairpin Motif in the Amyloid-& beta Peptide Is Important for Formation of Disease-Related Oligomers2023Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 145, nr 33, s. 18340-18354Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The amyloid-& beta;(A & beta;) peptide is associated with the developmentof Alzheimer's disease and is known to form highly neurotoxicprefibrillar oligomeric aggregates, which are difficult to study dueto their transient, low-abundance, and heterogeneous nature. To obtainhigh-resolution information about oligomer structure and dynamicsas well as relative populations of assembly states, we here employa combination of native ion mobility mass spectrometry and moleculardynamics simulations. We find that the formation of A & beta; oligomersis dependent on the presence of a specific & beta;-hairpin motif inthe peptide sequence. Oligomers initially grow spherically but startto form extended linear aggregates at oligomeric states larger thanthose of the tetramer. The population of the extended oligomers couldbe notably increased by introducing an intramolecular disulfide bond,which prearranges the peptide in the hairpin conformation, therebypromoting oligomeric structures but preventing conversion into maturefibrils. Conversely, truncating one of the & beta;-strand-formingsegments of A & beta; decreased the hairpin propensity of the peptideand thus decreased the oligomer population, removed the formationof extended oligomers entirely, and decreased the aggregation propensityof the peptide. We thus propose that the observed extended oligomerstate is related to the formation of an antiparallel sheet state,which then nucleates into the amyloid state. These studies provideincreased mechanistic understanding of the earliest steps in A & beta;aggregation and suggest that inhibition of A & beta; folding into thehairpin conformation could be a viable strategy for reducing the amountof toxic oligomers.

  • 60.
    Kilk, Kalle
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi. University of Tartu, Estonia.
    Magzoub, Mazin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pooga, Margus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi. Estonian Biocenter, Estonia.
    Eriksson, L. E. Göran
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cellular internalization of a cargo complex with a novel peptide derived from the third helix of the islet-1 homeodomain: Comparison with the penetratin peptide2001Ingår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 12, nr 6, s. 911-916Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cellular translocation into a human Bowes melanoma cell line was investigated and compared for penetratin and pIsl, two peptides that correspond to the third helices of the related homeodomains, from the Antennapedia transcription factor of Drosophila and the rat insulin-1 gene enhancer protein, respectively. Both biotinylated peptides internalized into the cells with similar efficacy, yielding an analogous intracellular distribution. When a large cargo protein, 63 kDa avidin, was coupled to either peptide, efficient cellular uptake for both the peptide−protein complexes was observed. The interactions between each peptide and SDS micelles were studied by fluorescence spectroscopy and acrylamide quenching of the intrinsic tryptophan (Trp) fluorescence. Both peptides interacted strongly and almost identically with the membrane mimicking environment. Compared to penetratin, the new transport peptide pIsl has only one Trp residue, which simplifies the interpretation of the fluorescence spectra and in addition has a native Cys residue, which may be used for alternative coupling reactions of cargoes of different character.

  • 61. Kolberg, Matthias
    et al.
    Bleifuss, Günter
    Sjöberg, Britt-Marie
    Stockholms universitet.
    Gräslund, Astrid
    Stockholms universitet.
    Lubitz, Wolfgang
    Lendzian, Friedhelm
    Lassmann, Günter
    Generation and Electron Paramagnetic Resonance Spin Trapping Detection of Thinyl Radicals in Model Proteins and in the R1 Subunit of Escherichia coli2002Ingår i: Archives of Biochemistry & Biophysics, Vol. 397, s. 57-68Artikel i tidskrift (Refereegranskat)
  • 62. Kolberg, Matthias
    et al.
    Logan, Derek
    Bleifuss, Günther
    Pötsch, Stephan
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Lubitz, Wolfgang
    Lassmann, Günter
    Lendzian, Friedhelm
    A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies.2005Ingår i: J Biol Chem, ISSN 0021-9258, Vol. 280, nr 12, s. 11233-46Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.

  • 63. Kolberg, Matthis
    et al.
    Bleifuss, Günter
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sjöberg, Britt-Marie
    Institutionen för molekylärbiologi och funktionsgenomik.
    Lubitz, Wolfgang
    Lendzian, Friedhelm
    Lassmann, Günter
    Protein thiyl directly observed by EPR spectroscopy2002Ingår i: Arcives of Biochemistry & Biophysics, Vol. 403, s. 141-144Artikel i tidskrift (Refereegranskat)
  • 64.
    Król, Sylwia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Österlund, Nicklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vosough, Faraz
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Barth, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ilag, Leopold Luna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Magzoub, Mazin
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mörman, Cecilia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The amyloid-inhibiting NCAM-PrP peptide targets Aβ peptide aggregation in membrane-mimetic environments2021Ingår i: iScience, E-ISSN 2589-0042 , Vol. 24, nr 8, artikel-id 102852Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Substantial research efforts have gone into elucidating the role of protein misfolding and self-assembly in the onset and progression of Alzheimer’s disease (AD). Aggregation of the Amyloid-β (Aβ) peptide into insoluble fibrils is closely associated with AD. Here, we use biophysical techniques to study a peptide-based approach to target Aβ amyloid aggregation. A peptide construct, NCAM-PrP, consists of a largely hydrophobic signal sequence linked to a positively charged hexapeptide. The NCAM-PrP peptide inhibits Aβ amyloid formation by forming aggregates which are unavailable for further amyloid aggregation. In a membrane-mimetic environment, Aβ and NCAM-PrP form specific heterooligomeric complexes, which are of lower aggregation states compared to Aβ homooligomers. The Aβ:NCAM-PrP interaction appears to take place on different aggregation states depending on the absence or presence of a membrane-mimicking environment. These insights can be useful for the development of potential future therapeutic strategies targeting Aβ at several aggregation states.

  • 65.
    Langel, Ülo
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cravatt, Benjamin F.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Land, Tiit
    Niessen, Sherry
    Zorko, Matjaz
    Introduction to peptides and proteins2010 (uppl. 1)Bok (Refereegranskat)
  • 66. Leidel, Nils
    et al.
    Popovic-Bijelic, Ana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Havelius, Kajsa G. V.
    Chernev, Petko
    Voevodskaya, Nina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Haumann, Michael
    High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases2012Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1817, nr 3, s. 430-444Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-la RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Cr R2, two different Mn(IV)Fe(III) site configurations were assigned either L4MnIV(mu O)(2)(FeL4)-L-III (metal-metal distance of similar to 2.75 angstrom, L = ligand) prevailing in metal-grown R2, or L4MnIV(mu O)(mu OH)(FeL4)-L-III (similar to 2.90 angstrom) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (similar to 2.55 angstrom) with a L4FeIV(mu O)(2)(FeL3)-L-III core structure. Several Mn,Fe(III)Fe(III) (similar to 2.9-3.1 angstrom) and Mn,Fe(III)Fe(II) species (similar to 3.3-3.4 angstrom) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit RI. In Ct R2, the protonation of a bridging oxide in the Mn-IV(mu O)(mu OH)Fe-III core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.

  • 67. Lendel, Christofer
    et al.
    Bolognesi, Benedetta
    Wahlström, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dobson, Christopher M.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Detergent-like interaction of Congo red with the amyloid beta peptide2010Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, nr 7, s. 1358-1360Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Accumulating evidence links prefibrillar oligomeric species of the amyloid beta peptide (Abeta) to cellular toxicity in Alzheimer's disease, potentially via disruption of biological membranes. Congo red (CR) affects protein aggregation. It is known to self-associate into micelle-like assemblies but still reduces the toxicity of Abeta aggregates in cell cultures and model organisms. We show here that CR interacts with Abeta(1-40) in a manner similar to that of anionic detergents. Although CR promotes beta sheet formation and peptide aggregation, it may also solubilize toxic protein species, making them less harmful to critical cellular components and thereby reducing amyloid toxicity.

  • 68. Lendzian, F.
    et al.
    Voevodskaya, Nina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Galander, M.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The high-valent Fe(III)Fe(IV) center in class Ic ribonucleotide reductase of chlamydia trachomatis: EPR and ENDOR studies2007Konferensbidrag (Övrig (populärvetenskap, debatt, mm))
  • 69. Leshem, Guy
    et al.
    Richman, Michal
    Lisniansky, Elvira
    Antman-Passig, Merav
    Habashi, Maram
    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.
    Rahimipour, Shai
    Photoactive chlorin e6 is a multifunctional modulator of amyloid-β aggregation and toxicity via specific interactions with its histidine residues2019Ingår i: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 10, nr 1, s. 208-217Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The self-assembly of A to -sheet-rich neurotoxic oligomers is a main pathological event leading to Alzheimer's disease (AD). Selective targeting of A oligomers without affecting other functional proteins is therefore an attractive approach to prevent the disease and its progression. In this study, we report that photodynamic treatment of A in the presence of catalytic amounts of chlorin e6 can selectively damage A and inhibit its aggregation and toxicity. Chlorin e6 also reversed the amyloid aggregation process in the dark by binding its soluble and low molecular weight oligomers, as shown by thioflavin T (ThT) fluorescence and photoinduced cross-linking of unmodified protein (PICUP) methods. Using HSQC NMR spectroscopy, ThT assays, amino acid analysis, SDS/PAGE, and EPR spectroscopy, we show that catalytic amounts of photoexcited chlorin e6 selectively damage the A histidine residues H6, H13, and H14, and induce A cross-linking by generating singlet oxygen. In contrast, photoexcited chlorin e6 was unable to cross-link ubiquitin and -synuclein, demonstrating its high selectivity for A. By binding to the A histidine residues, catalytic amounts of chlorin e6 can also inhibit the Cu2+-induced aggregation and toxicity in darkness, while at stoichiometric amounts it acts as a chelator to reduce the amount of free Cu2+. This study demonstrates the great potential of chlorin e6 as a multifunctional agent for treatment of AD, and shows that the three N-terminal A histidine residues are a suitable target for A-specific drugs.

  • 70.
    Lind, Jesper
    et al.
    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 Interactions of Dynorphins2006Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, s. 15931-15940Artikel i tidskrift (Refereegranskat)
  • 71. Lindgren, Joel
    et al.
    Segerfeldt, Patrik
    Sholts, Sabrina B.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Karlström, Amelie Eriksson
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Engineered non-fluorescent Affibody molecules facilitate studies of the amyloid-beta (A beta) peptide in monomeric form: Low pH was found to reduce A beta/Cu(II) binding affinity2013Ingår i: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 120, s. 18-23Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aggregation of amyloid-beta (A beta) peptides into oligomers and amyloid plaques in the human brain is considered a causative factor in Alzheimer's disease (AD). As metal ions are over-represented in AD patient brains, and as distinct A beta aggregation pathways in presence of Cu(II) have been demonstrated, metal binding to A beta likely affects AD progression. A beta aggregation is moreover pH-dependent, and AD appears to involve inflammatory conditions leading to physiological acidosis. Although metal binding specificity to A beta varies at different pH's, metal binding affinity to A beta has so far not been quantitatively investigated at sub-neutral pH levels. This may be explained by the difficulties involved in studying monomeric peptide properties under aggregation-promoting conditions. We have recently devised a modified Affibody molecule, Z(A beta 3)(12-58), that binds A beta with sub-nanomolar affinity, thereby locking the peptide in monomeric form without affecting the N-terminal region where metal ions bind. Here, we introduce non-fluorescent A beta-binding Affibody variants that keep A beta monomeric while only slightly affecting the A beta peptide's metal binding properties. Using fluorescence spectroscopy, we demonstrate that Cu(II)/A beta(1-40) binding is almost two orders of magnitude weaker at pH 5.0 (apparent K-D = 51 mu M) than at pH 7.3 (apparent K-D = 0.86 mu M). This effect is arguably caused by protonation of the histidines involved in the metal ligandation. Our results indicate that engineered variants of Affibody molecules are useful for studying metal-binding and other properties of monomeric A beta under various physiological conditions, which will improve our understanding of the molecular mechanisms involved in AD.

  • 72. Lindgren, Joel
    et al.
    Wahlström, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Markova, Natalia
    Ekblad, Caroline
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abrahmsen, Lars
    Eriksson Karlström, Amelie
    Wärmlander, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    N-terminal engineering of amyloid-β-binding Affibody molecules yields improved chemical synthesis and higher binding affinity2010Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, nr 12, s. 2319-2329Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The aggregation of amyloid-beta (A beta) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease Molecules binding with high affinity and selectivity to A beta-peptides are important tools for investigating the aggregation process An A beta-binding Affibody molecule, Z(A beta 3), has earlier been selected by phage display and shown to bind A beta(1-40) with nanomolar affinity and to inhibit A beta-peptide aggregation In this study, we create truncated functional versions of the Z(A beta 3) Affibody molecule better suited for chemical synthesis production Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges The N-terminally truncated Affibody molecules Z(A beta 3)(12-58), Z(A beta 3)(15-58), and Z(A beta 3)(18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule Z(A beta 3)(1-58) Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, Z(A beta 3)(18-58), exhibited complete loss of binding to the A beta(1-40)-peptide, while the Z(A beta 3)(12-58) and Z(A beta 3)(15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the A beta(1-40)-peptide compared to the full-length Affibody molecule Nuclear magnetic resonance spectroscopy showed that the structure of A beta(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the A beta(1-40)-peptide in complex with the full-length Z(A beta 3) Affibody molecule This indicates that the N-terminally truncated Affibody molecules Z(A beta 3)(12-58) and Z(A beta 3)(15-58) are highly promising for further engineering and future use as binding agents to monomeric A beta(1-40)

  • 73. Liu, Chang
    et al.
    Henning-Knechtel, Anja
    Österlund, Nicklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wu, Jinming
    Wang, Guangshun
    Gräslund, Rut Astrid Olivia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kirmizialtin, Serdal
    Luo, Jinghui
    Oligomer Dynamics of LL-37 Truncated Fragments Probed by α-Hemolysin Pore and Molecular Simulations2023Ingår i: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 19, nr 37, artikel-id 2206232Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Oligomerization of antimicrobial peptides (AMPs) is critical in their effects on pathogens. LL-37 and its truncated fragments are widely investigated regarding their structures, antimicrobial activities, and application, such as developing new antibiotics. Due to the small size and weak intermolecular interactions of LL-37 fragments, it is still elusive to establish the relationship between oligomeric states and antimicrobial activities. Here, an α-hemolysin nanopore, mass spectrometry (MS), and molecular dynamic (MD) simulations are used to characterize the oligomeric states of two LL-37 fragments. Nanopore studies provide evidence of trapping events related to the oligomer formation and provide further details on their stabilities, which are confirmed by MS and MD simulations. Furthermore, simulation results reveal the molecular basis of oligomer dynamics and states of LL-37 fragments. This work provides unique insights into the relationship between the oligomer dynamics of AMPs and their antimicrobial activities at the single-molecule level. The study demonstrates how integrating methods allows deciphering single molecule level understanding from nanopore sensing approaches. 

  • 74. Liu, Lin
    et al.
    Dong, Xin
    Liu, Yichang
    Österlund, Nicklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Carloni, Paolo
    Li, Jinyu
    Role of hydrophobic residues for the gaseous formation of helical motifs2019Ingår i: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 55, nr 35, s. 5147-5150Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The secondary structure content of proteins and their complexes may change significantly on passing from aqueous solution to the gas phase (as in mass spectrometry experiments). In this work, we investigate the impact of hydrophobic residues on the formation of the secondary structure of a real protein complex in the gas phase. We focus on a well-studied protein complex, the amyloid- (1-40) dimer (2A). Molecular dynamics simulations reproduce the results of ion mobility-mass spectrometry experiments. In addition, a helix (not present in the solution) is identified involving (19)FFAED(23), consistent with infrared spectroscopy data on an A segment. Our simulations further point to the role of hydrophobic residues in the formation of helical motifs - hydrophobic sidechains shield helices from being approached by residues that carry hydrogen bond sites. In particular, two hydrophobic phenylalanine residues, F19 and F20, play an important role for the helix, which is induced in the gas phase in spite of the presence of two carboxyl-containing residues.

  • 75.
    Lundberg, Pontus
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi.
    Magzoub, Mazin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindberg, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi.
    Jarvet, Jüri
    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 och neurotoxikologi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cell membrane translocation of the N-terminal (1-28) part of the prion protein2002Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 299, nr 1, s. 85-90Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The N-terminal (1-28) part of the mouse prion protein (PrP) is a cell penetrating peptide, capable of transporting large hydrophilic cargoes through a cell membrane. Confocal fluorescence microscopy shows that it transports the protein avidin (67 kDa) into several cell lines. The (1-28) peptide has a strong tendency for aggregation and P-structure formation, particularly in interaction with negatively charged phospholipid membranes. The findings have implications for how prion proteins with uncleaved signal peptides in the N-termini may enter into cells, which is important for infection. The secondary structure conversion into beta-structure may be relevant as a seed for the conversion into the scrapie (PrPSc) form of the protein and its arnyloidic transformation.

  • 76.
    Luo, Jinghui
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ribonucleotide reductase inhibition by p-alkoxyphenols studied by molecular docking and molecular dynamics simulations.2011Ingår i: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 516, nr 1, s. 29-34Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase (RNR) is necessary for production of the precursor deoxyribonucleotides for DNA synthesis. Class Ia RNR functions via a stable free radical in one of the two components protein R2. The enzyme mechanism involves long range (proton coupled) electron transfer between protein R1 and the tyrosyl radical in protein R2. Earlier experimental studies showed that p-alkoxyphenols inhibit RNR. Here, molecular docking and molecular dynamics simulations involving protein R2 suggest an inhibition mechanism for p-alkoxyphenols . A low energy binding pocket is identified in protein R2. The preferred configuration provides a structural basis explaining their specific binding to the Escherichia coli and mouse R2 proteins. Trp48 (E. coli numbering), on the electron transfer pathway, is involved in the interactions with the inhibitors. The relative order of the binding energies calculated for the phenol derivatives to protein R2 is correlated with earlier experimental data on inhibition efficiency, in turn related to increasing size of the hydrophobic alkyl substituents. Using the configuration identified by molecular docking as a starting point for molecular dynamics simulations, we find that the p-allyloxyphenol interrupts the catalytic electron transfer pathway of the R2 protein by forming hydrogen bonds with Trp48 and Asp237, thus explaining the inhibitory activity of p-alkoxyphenols.

  • 77.
    Luo, Jinghui
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Maréchal, Jean-Didier
    Wärmländer, Sebastian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Perálvarez-Marín, Alex
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    In silico analysis of the apolipoprotein E and the amyloid beta peptide interaction: misfolding induced by frustration of the salt bridge network2010Ingår i: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 6, nr 2, s. e1000663-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The relationship between Apolipoprotein E (ApoE) and the aggregation processes of the amyloid beta (A beta) peptide has been shown to be crucial for Alzheimer's disease (AD). The presence of the ApoE4 isoform is considered to be a contributing risk factor for AD. However, the detailed molecular properties of ApoE4 interacting with the A beta peptide are unknown, although various mechanisms have been proposed to explain the physiological and pathological role of this relationship. Here, computer simulations have been used to investigate the process of A beta interaction with the N-terminal domain of the human ApoE isoforms (ApoE2, ApoE3 and ApoE4). Molecular docking combined with molecular dynamics simulations have been undertaken to determine the A beta peptide binding sites and the relative stability of binding to each of the ApoE isoforms. Our results show that from the several ApoE isoforms investigated, only ApoE4 presents a misfolded intermediate when bound to A beta. Moreover, the initial alpha-helix used as the A beta peptide model structure also becomes unstructured due to the interaction with ApoE4. These structural changes appear to be related to a rearrangement of the salt bridge network in ApoE4, for which we propose a model. It seems plausible that ApoE4 in its partially unfolded state is incapable of performing the clearance of A beta, thereby promoting amyloid forming processes. Hence, the proposed model can be used to identify potential drug binding sites in the ApoE4-A beta complex, where the interaction between the two molecules can be inhibited.

  • 78. Luo, Jinghui
    et al.
    Mohammed, Inayathulla
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hiruma, Yoshitaka
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abrahams, Jan Pieter
    Endogenous Polyamines Reduce the Toxicity of Soluble A beta Peptide Aggregates Associated with Alzheimer's Disease2014Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, nr 6, s. 1985-1991Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Polyamines promote the formation of the A beta peptide amyloid fibers that are a hallmark of Alzheimer's disease. Here we show that polyamines interact with nonaggregated A beta peptides, thereby reducing the peptide's hydrophobic surface. We characterized the associated conformational change through NMR titrations and molecular dynamics simulations. We found that even low concentrations of spermine, sperimidine, and putrescine fully protected SH-SY5Y (a neuronal cell model) against the most toxic conformational species of AA even at an A beta oligomer concentration that would otherwise kill half of the cells or even more. These observations lead us to conclude that polyamines interfere with the more toxic prefibrillar conformations and might protect cells by promoting the structural transition of A beta toward its less toxic fibrillar state that we reported previously. Since polyamines are present in brain fluid at the concentrations where we observed all these effects, their activity needs to be taken into account in understanding the molecular processes related to the development of Alzheimer's disease.

  • 79. Luo, Jinghui
    et al.
    Otero, José M
    Yu, Chien-Hung
    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.
    Overhand, Mark
    Abrahams, Jan Pieter
    Inhibiting and Reversing Amyloid-β Peptide (1-40) Fibril Formation with Gramicidin S and Engineered Analogues2013Ingår i: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 19, nr 51, s. 17338-17348Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In Alzheimer's disease, amyloid-β (Aβ) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aβ peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aβ amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a β-sheet conformation, binds to the Aβ peptide hairpin-stacked fibril through β-sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four-times higher than that of the natural product was identified.

  • 80. Luo, Jinghui
    et al.
    Wärmlander, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abrahams, Jan Pieter
    Alzheimer Peptides Aggregate into Transient Nanoglobules That Nucleate Fibrils2014Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 53, nr 40, s. 6302-6308Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Protein/peptide oligomerization, cross-beta strand fibrillation, and amyloid deposition play a critical role in many diseases, but despite extensive biophysical characterization, the structural and dynamic details of oligomerization and fibrillation of amyloidic peptides/proteins remain to be fully clarified. Here, we simultaneously monitored the atomic, molecular, and mesoscopic states of aggregating Alzheimer's amyloid beta (A beta) peptides over time, using a slow aggregation protocol and a fast aggregation protocol, and determined the cytotoxicity of the intermediate states. We show that in the early stage of fast fibrillation (the lag phase) the A beta peptides coalesced into apparently unstructured globules (15-200 nm in diameter), which slowly grew larger. Then a sharp transition occurred, characterized by the first appearance of single fibrillar structures of approximately >= 100 nm. These fibrils emerged from the globules. Simultaneously, an increase was observed for the cross-beta strand conformation that is characteristic of the fibrils that constitute mature amyloid. The number and size of single fibrils rapidly increased. Eventually, the fibrils coalesced into mature amyloid. Samples from the early lag phase of slow fibrillation conditions were especially toxic to cells, and this toxicity sharply decreased when fibrils formed and matured into amyloid. Our results suggest that the formation of fibrils may protect cells by reducing the toxic structures that appear in the early lag phase of fibrillation.

  • 81. Luo, Jinghui
    et al.
    Wärmlander, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Yu, Chien-Hung
    Muhammad, Kamran
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abrahams, Jan Pieter
    The A beta peptide forms non-amyloid fibrils in the presence of carbon nanotubes2014Ingår i: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 6, nr 12, s. 6720-6726Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Carbon nanotubes have specific properties that make them potentially useful in biomedicine and biotechnology. However, carbon nanotubes may themselves be toxic, making it imperative to understand how carbon nanotubes interact with biomolecules such as proteins. Here, we used NMR, CD, and ThT/fluorescence spectroscopy together with AFM imaging to study pH-dependent molecular interactions between single walled carbon nanotubes (SWNTs) and the amyloid-beta (A beta) peptide. The aggregation of the A beta peptide, first into oligomers and later into amyloid fibrils, is considered to be the toxic mechanism behind Alzheimer's disease. We found that SWNTs direct the A beta peptides to form a new class of beta-sheet-rich yet non-amyloid fibrils.

  • 82. Luo, Jinghui
    et al.
    Wärmländer, Sebastian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abrahams, Jan Pieter
    Human lysozyme inhibits the in vitro aggregation of A beta peptides, which in vivo are associated with Alzheimer's disease2013Ingår i: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 49, nr 58, s. 6507-6509Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alzheimer's disease is a neurodegenerative disorder characterized by accumulation of A beta peptide aggregates in the brain. Using ThT fluorescence assays, AFM imaging, NMR and CD spectroscopy, and MD modeling we show that lysozyme - a hydrolytic enzyme abundant in human secretions - completely inhibits the aggregation of A beta peptides at equimolar lysozyme : A beta peptide ratios.

  • 83. Luo, Jinghui
    et al.
    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.
    Abrahams, Jan Pieter
    Cross-interactions between the Alzheimer Disease Amyloid-beta Peptide and Other Amyloid Proteins: A Further Aspect of the Amyloid Cascade Hypothesis2016Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, nr 32, s. 16485-16493Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Many protein folding diseases are intimately associated with accumulation of amyloid aggregates. The amyloid materials formed by different proteins/peptides share many structural similarities, despite sometimes large amino acid sequence differences. Some amyloid diseases constitute risk factors for others, and the progression of one amyloid disease may affect the progression of another. These connections are arguably related to amyloid aggregates of one protein being able to directly nucleate amyloid formation of another, different protein: the amyloid cross-interaction. Here, we discuss such cross-interactions between the Alzheimer disease amyloid-beta (A beta) peptide and other amyloid proteins in the context of what is known from in vitro and in vivo experiments, and of what might be learned from clinical studies. The aim is to clarify potential molecular associations between different amyloid diseases. We argue that the amyloid cascade hypothesis in Alzheimer disease should be expanded to include cross-interactions between A beta and other amyloid proteins.

  • 84. Luo, Jinghui
    et al.
    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.
    Abrahams, Jan Pieter
    Non-chaperone Proteins Can Inhibit Aggregation and Cytotoxicity of Alzheimer Amyloid beta Peptide2014Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, nr 40, s. 27766-27775Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: A amyloid formation is associated with Alzheimer disease. Results: Non-chaperone proteins prevent amyloid formation and reduce the cytotoxicity of the A peptide. Conclusion: Non-chaperone proteins may affect the onset and development of Alzheimer disease by interfering with A peptide aggregation. Significance: Non-chaperone proteins can function as a chaperone protein to regulate the pathway of the A fibrillation in proteostasis providing a new strategy in the treatment of Alzheimer disease. Many factors are known to influence the oligomerization, fibrillation, and amyloid formation of the A peptide that is associated with Alzheimer disease. Other proteins that are present when A peptides deposit in vivo are likely to have an effect on these aggregation processes. To separate specific versus broad spectrum effects of proteins on A aggregation, we tested a series of proteins not reported to have chaperone activity: catalase, pyruvate kinase, albumin, lysozyme, -lactalbumin, and -lactoglobulin. All tested proteins suppressed the fibrillation of Alzheimer A(1-40) peptide at substoichiometric ratios, albeit some more effectively than others. All proteins bound non-specifically to A, stabilized its random coils, and reduced its cytotoxicity. Surprisingly, pyruvate kinase and catalase were at least as effective as known chaperones in inhibiting A aggregation. We propose general mechanisms for the broad-spectrum inhibition A fibrillation by proteins. The mechanisms we discuss are significant for prognostics and perhaps even for prevention and treatment of Alzheimer disease.

  • 85. Luo, Jinghui
    et al.
    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.
    Abrahams, Jan Pieter
    Reciprocal Molecular Interactions between the A beta Peptide Linked to Alzheimer's Disease and Insulin Linked to Diabetes Mellitus Type II2016Ingår i: ACS Chemical Neuroscience, E-ISSN 1948-7193, Vol. 7, nr 3, s. 269-274Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Clinical studies indicate diabetes mellitus type II (DM) doubles the risk that a patient will also develop Alzheimer's disease (AD). DM is caused by insulin resistance and a relative lack of active insulin. AD is characterized by the deposition of amyloid beta (A beta) peptide fibrils. Prior to fibrillating, A beta forms intermediate, prefibrillar oligomers, which are more cytotoxic than the mature A beta fibrils. Insulin can also form amyloid fibrils. In vivo studies have revealed that insulin promotes the production of A beta, and that soluble A beta competes with insulin for the insulin receptor. Here, we report that monomeric insulin interacted with soluble A beta and that both molecules reciprocally slowed down the aggregation kinetics of the other. Prefibrillar oligomers of A beta that eventually formed in the presence of insulin were less cytotoxic than A beta oligomers formed in the absence of insulin. Mature A beta fibrils induced fibrillation of soluble insulin, but insulin aggregates did not promote A beta fibrillation. Our study indicates that direct molecular interactions between insulin and A beta may contribute to the strong link between DM and AD.

  • 86. Luo, Jinghui
    et al.
    Yu, Chien-Hung
    Yu, Huixin
    Borstnar, Rok
    Kamerlin, Shina C. L.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abrahams, Jan Pieter
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cellular Polyamines Promote Amyloid-Beta (A beta) Peptide Fibrillation and Modulate the Aggregation Pathways2013Ingår i: ACS Chemical Neuroscience, E-ISSN 1948-7193, Vol. 4, nr 3, s. 454-462Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The cellular polyamines spermine, spermidine, and their metabolic precursor putrescine, have long been associated with cell-growth, tumor-related gene regulations, and Alzheimer's disease. Here, we show by in vitro spectroscopy and AFM imaging, that these molecules promote aggregation of amyloid-beta (A beta) peptides into fibrils and modulate the aggregation pathways. NMR measurements showed that the three polyamines share a similar binding mode to monomeric A beta(1-40) peptide. Kinetic ThT studies showed that already very low polyamine concentrations promote amyloid formation: addition of 10 mu M spermine (normal intracellular concentration is similar to 1 mM) significantly decreased the lag and transition times of the aggregation process. Spermidine and putrescine additions yielded similar but weaker effects. CD measurements demonstrated that the three polyamines induce different aggregation pathways, involving different forms of induced secondary structure. This is supported by AFM images showing that the three polyamines induce A beta(1-40) aggregates with different morphologies. The results reinforce the notion that designing suitable ligands which modulate the aggregation of A beta peptides toward minimally toxic pathways may be a possible therapeutic strategy for Alzheimer's disease.

  • 87.
    Löfgren, Kajsa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wahlström, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bedecs, Katarina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Antiprion properties of prion protein-derived cell-penetrating peptides2008Ingår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 22, nr 7, s. 2177-2184Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In prion diseases, the cellular prion protein (PrPC) becomes misfolded into the pathogenic scrapie isoform (PrPSc) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1–1) and scrapie-infected (ScGT1–1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrPC or PrPSc. Treatment with the prion protein-derived CPPs mouse mPrP1–28 or bovine bPrP1–30 significantly reduced PrPSc levels in prion-infected cells but had no effect on PrPC levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1–1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP23–28, mPrP19–30, or mPrP23–50) had no effect on PrPSc levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrPSc and disables formation of prions.—Löfgren, K., Wahlström, A., Lundberg, P., Langel, U., Gräslund, A., and Bedecs, K. Antiprion properties of prion protein-derived cell-penetrating peptides.

  • 88.
    Löfgren Söderberg, Kajsa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mechanisms of prion antagonization by PrP-derived cell-penetrating peptidesManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Cell penetrating peptides derived from the prion protein N-terminus (PrP-CPPs) reduce PrPSc levels in prion-infected neuronal cell cultures (1). The PrP-CPPs consist of the hydrophobic PrP signal sequence followed by a basic segment (KKRPKP) and enter cells through raft-dependent macropinocytosis. To decipher the PrP-CPP anti-prion mechanism, different peptide constructs were analyzed for effects on PrPSc levels in GT1-1 neuronal cell cultures infected with either prion strain RML or 22L. For both strains, the PrP-CPPs antagonized the infection, but RML and 22L-infections differed in sensitivity to the PrP-CPP anti-prion effect. We also show that the effect on PrPSc levels does not depend on peptide interaction with any chiral receptor. The signal sequence segment of the PrP-CPPs promotes a specific positioning within the cell where conversion may occur, as signal sequence segment shortening or targeting of the KKRPKP-motif into alternative sub-cellular compartments disrupts the peptide anti-prion effect. Defining the anti-prion mechanism of PrP-CPPs is a matter of establishing how the peptides connect to the prion replicative interface. As the conversion process is poorly understood, the PrP-CPPs represent useful tools to outline the sub-cellular context of prion propagation.

  • 89.
    Löfgren Söderberg, Kajsa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Targeting prion propagation using peptide constructs with signal sequence motifs2014Ingår i: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 564, s. 254-261Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Synthetic peptides with sequences derived from the cellular prion protein (PrPc) unprocessed N-terminus are able to counteract the propagation of proteinase K resistant prions (PrPRes, indicating the presence of the prion isoform of the prion protein) in cell cultures (Lofgren et al., 2008). The anti-prion peptides have characteristics like cell penetrating peptides (CPPs) and consist of the prion protein hydrophobic signal sequence followed by a polycationic motif (residues KKRPKP), in mouse PrPc corresponding to residues 1-28. Here we analyze the sequence elements required for the anti-prion effect of KKRPKP-conjugates. Neuronal GT1-1 cells were infected with either prion strain RML or 22L Variable peptide constructs originating from the mPrP(1-28) sequence were analyzed for anti-prion effects, measured as disappearance of proteinase K resistant prions (PrPRes) in the infected cell cultures. We find that even a 5 amino acid N-terminal shortening of the signal peptide abolishes the anti-prion effect. We show that the signal peptide from PrPc can be replaced with the signal peptide from the Neural cell adhesion molecule-1; NCAMl(1-19), with a retained capacity to reduce PrPRes levels. The anti-prion effect is lost if the polycationic N-terminal PrPc-motif is conjugated to any conventional CPP, such as TAT(48-60), transportan-10 or penetratin. We propose a mechanism by which a signal peptide from a secretory or cell surface protein acts to promote the transport of a prion-binding polycationic PrPc-motif to a subcellular location where prion conversion occurs (most likely the Endosome Recycling Compartment), thereby targeting prion propagation.

  • 90.
    Madani, Fatemeh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abdo, Rania
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hirose, Hisaaki
    Futaki, Shiroh
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient2013Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, nr 4, s. 1198-1204Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

  • 91. Madani, Fatemeh
    et al.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Investigating Membrane Interactions and Structures of CPPs2015Ingår i: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, New York: Springer, 2015, Vol. 1324, s. 73-87Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Despite many studies made on cell-penetrating peptides (CPPs), the mechanism of their cellular uptake and endosomal escape has not been completely resolved. This is even more unclear when the CPP is bound either covalently or non-covalently to the cargo molecules. To answer remaining questions, we require a combination of different methods, model systems, and experiments since there is no single method which could give a complete answer to all questions. Biophysical investigations of CPPs have a significant impact on CPP research considering their molecular mechanisms of action. In this chapter, we present different membrane model systems suitable for biophysical studies as well as the basic practical aspects underlying several common biophysical methods and experiments. The methods include fluorescence spectroscopy, circular dichroism spectroscopy, and dynamic light scattering and concern peptide-membrane interactions and vesicle model membrane leakage. We have also described the potential and limitations of biophysical studies on the CPP-membrane interactions and their impact on our understanding of how CPPs mediate the transport of cargoes into living cells.

  • 92. Madani, Fatemeh
    et al.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane Molecular Interactions and Induced Structures of CPPs2022Ingår i: Cell Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, New York: Humana Press, 2022, 3, s. 153-165Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Cell penetrating peptides (CPPs) are generally defined as short positively charged peptides, containing 5–30 amino acids. Based on their physicochemical properties, they are classified as three main groups, namely hydrophobic, amphipathic, and hydrophilic. They are capable of interacting with the cell membrane without inducing serious toxicity, and they can carry cargo molecules across the membrane. Cargo molecules could be different therapeutics which makes CPPs valuable in the field of drug delivery into living cells. Nowadays, CPPs are considered as potential parts of therapeutics against several diseases. Despite similarities in their primary structure, the interactions of CPPs with a cell membrane may vary a lot. This is even more complicated when the CPP is bound to the cargo molecule. The mechanism(s) of their cellular uptake and endosomal escape have not been completely resolved. Understanding the mechanism of membrane interaction will help us designing a CPP with enhanced, selective cargo delivery, hopefully resulting in better disease treatments. So far energy independent direct membrane penetration and energy-dependent endocytosis have been suggested as two main mechanisms of cellular entry for CPPs, and both may be applicable for the same CPP-complex, depending on the conditions. In order to understand which mechanism is associated with a particular CPP’s cellular uptake in a particular cell (sometimes including endosomal escape), different biological and biophysical methods and strategies have been applied. In this chapter, we will address several biophysical methods, such as fluorescence spectroscopy, circular dichroism (CD) spectroscopy, dynamic light scattering, and NMR. We also review different membrane model systems which are suitable for the biophysical studies. These include large unilamellar phospholipid vesicles (LUVs), which are the most commonly used in the lipid–peptide interaction studies. Detergent micelles and mixed micelles (bicelles) are also suitable membrane model systems, particularly in high-resolution NMR studies. 

  • 93.
    Madani, Fatemeh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Damberg, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Adams, Stephen
    Tsien, Roger
    Gräslund, Astrid O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hairpin Structure of a Biarsenical−Tetracysteine Motif Determined by NMR Spectroscopy2009Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, nr 13, s. 4613-4615Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The biarsenical−tetracysteine motif is a useful tag for genetic labeling of proteins with small molecules in living cells. The present study concerns the structure of a 12 amino acid peptide FLNCCPGCCMEP bound to the fluorophore ReAsH based on resorufin. 1H NMR spectroscopy was used to determine the solution structure of the complex formed between the peptide and the ReAsH moiety. Structure calculations based on the NMR results showed that the backbone structure of the peptide is fairly well defined, with a hairpinlike turn, similar to a type-II β-turn, formed by the central CPGC segment. The most stable complex was formed when As2 was bonded to C4 and C5 and As1 to C8 and C9. Two clear NOESY cross-peaks between the Phe1 side chain and ReAsH confirmed the close positioning of the phenyl ring of Phe1 and ReAsH. Phe1 was found to have an edge−face geometry relative to ReAsH. The close interaction between Phe1 and ReAsH may be highly significant for the fluorescence properties of the ReAsH complex.

  • 94.
    Madani, Fatemeh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Futaki, Shiroh
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mechanisms of Cellular Uptake of Cell-Penetrating Peptides2011Ingår i: Journal of Biophysics, ISSN 1687-8000, E-ISSN 1687-8019, artikel-id 414729Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Recently, much attention has been given to the problem of drug delivery through the cell-membrane in order to treat and manage several diseases. The discovery of cell penetrating peptides (CPPs) represents a major breakthrough for the transport of large-cargo molecules that may be useful in clinical applications. CPPs are rich in basic amino acids such as arginine and lysine and are able to translocate over membranes and gain access to the cell interior. They can deliver large-cargo molecules, such as oligonucleotides, into cells. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms, a subject still under debate. Unresolved questions include the detailed molecular uptake mechanism(s), reasons for cell toxicity, and the delivery efficiency of CPPs for different cargoes. Here, we give a review focused on uptake mechanisms used by CPPs for membrane translocation and certain experimental factors that affect the mechanism(s).

  • 95.
    Madani, Fatemeh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Perálvarez-Marín, Alex
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport Across Phospholipid Membranes Induced by a Proton Gradient2011Ingår i: Journal of drug delivery, ISSN 2090-3022, Vol. 2011, s. 897592-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Detergent-mediated reconstitution of bacteriorhodopsin (BR) into large unilamellar vesicles (LUVs) was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG) as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane.

  • 96.
    Madani, Fatemeh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Taqi, Malik Mumtaz
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Verbeek, Dineke S.
    Bakalkin, Georgy
    Gräslund, Astrid