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  • 51. Leidel, Nils
    et al.
    Popovic-Bijelic, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Havelius, Kajsa G. V.
    Chernev, Petko
    Voevodskaya, Nina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haumann, Michael
    High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases2012In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1817, no 3, p. 430-444Article in journal (Refereed)
    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.

  • 52. Lendel, Christofer
    et al.
    Bolognesi, Benedetta
    Wahlström, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dobson, Christopher M.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Detergent-like interaction of Congo red with the amyloid beta peptide2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 7, p. 1358-1360Article in journal (Refereed)
    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.

  • 53. Lendzian, F.
    et al.
    Voevodskaya, Nina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Galander, M.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The high-valent Fe(III)Fe(IV) center in class Ic ribonucleotide reductase of chlamydia trachomatis: EPR and ENDOR studies2007Conference paper (Other (popular science, discussion, etc.))
  • 54.
    Lind, Jesper
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane Interactions of Dynorphins2006In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, p. 15931-15940Article in journal (Refereed)
  • 55. Lindgren, Joel
    et al.
    Segerfeldt, Patrik
    Sholts, Sabrina B.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karlström, Amelie Eriksson
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    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 affinity2013In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 120, p. 18-23Article in journal (Refereed)
    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.

  • 56. Lindgren, Joel
    et al.
    Wahlström, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Markova, Natalia
    Ekblad, Caroline
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahmsen, Lars
    Eriksson Karlström, Amelie
    Wärmlander, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    N-terminal engineering of amyloid-β-binding Affibody molecules yields improved chemical synthesis and higher binding affinity2010In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, no 12, p. 2319-2329Article in journal (Refereed)
    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)

  • 57.
    Lundberg, Pontus
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Magzoub, Mazin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindberg, Mattias
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Jarvet, Jüri
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, L. E. Göran
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cell membrane translocation of the N-terminal (1-28) part of the prion protein2002In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 299, no 1, p. 85-90Article in journal (Refereed)
    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.

  • 58.
    Luo, Jinghui
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ribonucleotide reductase inhibition by p-alkoxyphenols studied by molecular docking and molecular dynamics simulations.2011In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 516, no 1, p. 29-34Article in journal (Refereed)
    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.

  • 59.
    Luo, Jinghui
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Maréchal, Jean-Didier
    Wärmländer, Sebastian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Perálvarez-Marín, Alex
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    In silico analysis of the apolipoprotein E and the amyloid beta peptide interaction: misfolding induced by frustration of the salt bridge network2010In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 6, no 2, p. e1000663-Article in journal (Refereed)
    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.

  • 60. Luo, Jinghui
    et al.
    Mohammed, Inayathulla
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hiruma, Yoshitaka
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahams, Jan Pieter
    Endogenous Polyamines Reduce the Toxicity of Soluble A beta Peptide Aggregates Associated with Alzheimer's Disease2014In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, no 6, p. 1985-1991Article in journal (Refereed)
    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.

  • 61. Luo, Jinghui
    et al.
    Otero, José M
    Yu, Chien-Hung
    Wärmländer, Sebastian K T S
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Overhand, Mark
    Abrahams, Jan Pieter
    Inhibiting and Reversing Amyloid-β Peptide (1-40) Fibril Formation with Gramicidin S and Engineered Analogues2013In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 19, no 51, p. 17338-17348Article in journal (Refereed)
    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.

  • 62. Luo, Jinghui
    et al.
    Wärmlander, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahams, Jan Pieter
    Alzheimer Peptides Aggregate into Transient Nanoglobules That Nucleate Fibrils2014In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 53, no 40, p. 6302-6308Article in journal (Refereed)
    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.

  • 63. Luo, Jinghui
    et al.
    Wärmlander, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Yu, Chien-Hung
    Muhammad, Kamran
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahams, Jan Pieter
    The A beta peptide forms non-amyloid fibrils in the presence of carbon nanotubes2014In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 6, no 12, p. 6720-6726Article in journal (Refereed)
    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.

  • 64. Luo, Jinghui
    et al.
    Wärmländer, Sebastian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahams, Jan Pieter
    Human lysozyme inhibits the in vitro aggregation of A beta peptides, which in vivo are associated with Alzheimer's disease2013In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 49, no 58, p. 6507-6509Article in journal (Refereed)
    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.

  • 65. Luo, Jinghui
    et al.
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahams, Jan Pieter
    Cross-interactions between the Alzheimer Disease Amyloid-beta Peptide and Other Amyloid Proteins: A Further Aspect of the Amyloid Cascade Hypothesis2016In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 32, p. 16485-16493Article, review/survey (Refereed)
    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.

  • 66. Luo, Jinghui
    et al.
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahams, Jan Pieter
    Non-chaperone Proteins Can Inhibit Aggregation and Cytotoxicity of Alzheimer Amyloid beta Peptide2014In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 40, p. 27766-27775Article in journal (Refereed)
    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.

  • 67. Luo, Jinghui
    et al.
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahams, Jan Pieter
    Reciprocal Molecular Interactions between the A beta Peptide Linked to Alzheimer's Disease and Insulin Linked to Diabetes Mellitus Type II2016In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 7, no 3, p. 269-274Article in journal (Refereed)
    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.

  • 68. Luo, Jinghui
    et al.
    Yu, Chien-Hung
    Yu, Huixin
    Borstnar, Rok
    Kamerlin, Shina C. L.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahams, Jan Pieter
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cellular Polyamines Promote Amyloid-Beta (A beta) Peptide Fibrillation and Modulate the Aggregation Pathways2013In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 4, no 3, p. 454-462Article in journal (Refereed)
    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.

  • 69.
    Löfgren, Kajsa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wahlström, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundberg, Pontus
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bedecs, Katarina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Antiprion properties of prion protein-derived cell-penetrating peptides2008In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 22, no 7, p. 2177-2184Article in journal (Refereed)
    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.

  • 70.
    Löfgren Söderberg, Kajsa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mechanisms of prion antagonization by PrP-derived cell-penetrating peptidesManuscript (preprint) (Other academic)
    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.

  • 71.
    Löfgren Söderberg, Kajsa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Targeting prion propagation using peptide constructs with signal sequence motifs2014In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 564, p. 254-261Article in journal (Refereed)
    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.

  • 72.
    Madani, Fatemeh
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abdo, Rania
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hirose, Hisaaki
    Futaki, Shiroh
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient2013In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 4, p. 1198-1204Article in journal (Refereed)
    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.

  • 73. Madani, Fatemeh
    et al.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Investigating Membrane Interactions and Structures of CPPs2015In: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, New York: Springer, 2015, Vol. 1324, p. 73-87Chapter in book (Refereed)
    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.

  • 74.
    Madani, Fatemeh
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lind, Jesper
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Damberg, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Adams, Stephen
    Tsien, Roger
    Gräslund, Astrid O.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hairpin Structure of a Biarsenical−Tetracysteine Motif Determined by NMR Spectroscopy2009In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 13, p. 4613-4615Article in journal (Refereed)
    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.

  • 75.
    Madani, Fatemeh
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Futaki, Shiroh
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mechanisms of Cellular Uptake of Cell-Penetrating Peptides2011In: Journal of Biophysics, ISSN 1687-8000, E-ISSN 1687-8019, article id 414729Article, review/survey (Refereed)
    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).

  • 76.
    Madani, Fatemeh
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Perálvarez-Marín, Alex
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport Across Phospholipid Membranes Induced by a Proton Gradient2011In: Journal of drug delivery, ISSN 2090-3022, Vol. 2011, p. 897592-Article in journal (Refereed)
    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.

  • 77.
    Madani, Fatemeh
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Taqi, Malik Mumtaz
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Verbeek, Dineke S.
    Bakalkin, Georgy
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Perturbations of model membranes induced by pathogenic dynorphin A mutants causing neurodegeneration in human brain2011In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 411, no 1, p. 111-114Article in journal (Refereed)
    Abstract [en]

    Several effects of the endogenous opioid peptide dynorphin A (Dyn A) are not mediated through the opioid receptors. These effects are generally excitatory, and result in cell loss and induction of chronic pain and paralysis. The mechanism(s) is not well defined but may involve formation of pores in cellular membranes. In the 17-amino acid peptide Dyn A we have recently identified L5S, R6W, and R9C mutations that cause the dominantly inherited neurodegenerative disorder Spinocerebellar ataxia type 23. To gain further insight into non-opioid neurodegenerative mechanism(s), we studied the perturbation effects on lipid bilayers of wild type Dyn A and its mutants in large unilamellar phospholipid vesicles encapsulating the fluorescent dye calcein. The peptides were found to induce calcein leakage from uncharged and negatively charged vesicles to different degrees, thus reflecting different membrane perturbation effects. The mutant Dyn A R6W was the most potent in producing leakage with negatively charged vesicles whereas Dyn A L5S was virtually inactive. The overall correlation between membrane perturbation and neurotoxic response [3] suggests that pathogenic Dyn A actions may be mediated through transient pore formation in lipid domains of the plasma membrane.

  • 78.
    Magzoub, Mazin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kilk, Kalle
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Eriksson, L. E. Göran
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles2001In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1512, no 1, p. 77-89Article in journal (Refereed)
    Abstract [en]

    Certain short peptides, which are able to translocate across cell membranes with a low lytic activity, can be useful as carriers (vectors) for hydrophilic molecules. We have studied three such cell penetrating peptides: pAntp (‘penetratin’), pIsl and transportan. pAntp and pIsl originate from the third helix of homeodomain proteins (Antennapedia and Isl-1, respectively). Transportan is a synthetic chimera (galanin and mastoparan). The peptides in the presence of various phospholipid vesicles (neutral and charged) and SDS micelles have been characterized by spectroscopic methods (fluorescence, EPR and CD). The dynamics of pAntp were monitored using an N-terminal spin label. In aqueous solution, the CD spectra of the three peptides show secondary structures dominated by random coil. With phospholipid vesicles, neutral as well as negatively charged, transportan gives up to 60% α-helix. pAntp and pIsl bind significantly only to negatively charged vesicles with an induction of around 60% β-sheet-like secondary structure. With all three peptides, SDS micelles stabilize a high degree of α-helical structure. We conclude that the exact nature of any secondary structure induced by the membrane model systems is not directly correlated with the common transport property of these translocating peptides.

  • 79.
    Magzoub, Mazin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Staffan, Sandgren
    Pontus, Lundberg
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Oglecka, Kamila
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lilja, Johanna
    Wittrup, Anders
    Eriksson, L. E. Göran
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Belting, Mattias
    Astrid, Gräslund
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 348, no 2, p. 379-385Article in journal (Refereed)
    Abstract [en]

    A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1–24) and a basic domain (KKRPKP, residues 25–30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp–DNA–gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide’s ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases.

  • 80. Mitsueda, Asako
    et al.
    Shimatani, Yuri
    Ito, Masahiro
    Ohgita, Takashi
    Yamada, Asako
    Hama, Susumu
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Harashima, Hideyoshi
    Nakase, Ikuhiko
    Futaki, Shiroh
    Kogure, Kentaro
    Development of a Novel Nanoparticle by Dual Modification With the Pluripotential Cell-Penetrating Peptide PepFect6 for Cellular Uptake, Endosomal Escape, and Decondensation of an siRNA Core Complex2013In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 100, no 6, p. 698-704Article in journal (Refereed)
    Abstract [en]

    Development of novel devices for effective nucleotide release from nanoparticles is required to improve the functionality of nonviral delivery systems, because decondensation of nucleotide/polycation complexes is considered as a key step for cytoplasmic delivery of nucleotides. Previously, PepFect6 (PF6) comprised chloroquine analog moieties and a stearylated cell-penetrating peptide to facilitate endosomal escape and cellular uptake, respectively, was developed as a device for efficient siRNA delivery. As PF6 contains bulky chloroquine analog moieties, the polyplexes are expected to be loose structure, which facilitates decondensation. In the present study, siRNA was electrostatically condensed by PF6, and the PF6/siRNA complexes were coated with lipid membranes. The surface of the nanoparticles encapsulating the PF6/siRNA core (PF6-NP) was modified with PF6 for endosomal escape (PF6/PF6-NP). The RNAi effect of PF6/PF6-NP was compared with those of stearylated cell-penetrating peptide octaarginine (R8)-modified PF6-NP, R8-modified nanoparticles encapsulating the R8/siRNA core (R8-NP) and PF6-modified R8-NP. Nanoparticles encapsulating the PF6 polyplex, especially PF/PF-NP, showed a significant knockdown effect on luciferase activity of B16-F1 cells stably expressing luciferase. siRNA was widely distributed within the cytoplasm after transfection of the nanoparticles encapsulating the PF6 polyplex, while siRNA encapsulated in the R8-presenting nanoparticles was localized within the nuclei. Thus, the siRNA distribution was dependent on the manner of peptide-modification. In conclusion, we have successfully developed PF6/PF6-NP exhibiting a potent RNAi effect resulting from high cellular uptake, efficient endosomal escape and decondensation of the polyplexes based on the multifunctional cell penetrating peptide PF6. PF6 is therefore a useful pluripotential device for siRNA delivery. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 698-704, 2013.

  • 81.
    Mäler, Lena
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    NMR studies of three-dimensional structure and positioning of CPPs in membrane model systems2011In: Methods in molecular biology (Clifton, N.J.), ISSN 1940-6029, Vol. 683, p. 57-67Article in journal (Refereed)
    Abstract [en]

    CPPs are generally short cationic peptides that have the capability to interact directly with membranes. Most CPPs attain a three-dimensional structure when interacting with bilayers, while they are more or less unstructured in aqueous solution. To understand the relationship between structure and the effect that CPPs have on membranes, it is of great importance to investigate CPPs with atomic resolution in a suitable membrane model. Nuclear magnetic resonance (NMR) is an excellent technique both for studying solution structures of peptides as well as for investigating their location within a model bilayer. This chapter outlines protocols for producing model membrane systems for NMR investigations as well as the basic NMR tools for determining the three-dimensional structure of CPPs and for investigating the details in lipid-peptide interactions, i.e., the localization of the CPP in the bilayer.

  • 82. Nakase, Ikuhiko
    et al.
    Akita, Hidetaka
    Kogure, Kentaro
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Harashima, Hideyoshi
    Futaki, Shiroh
    Efficient Intracellular Delivery of Nucleic Acid Pharmaceuticals Using Cell-Penetrating Peptides2012In: Accounts of Chemical Research, ISSN 0001-4842, E-ISSN 1520-4898, Vol. 45, no 7, p. 1132-1139Article, review/survey (Refereed)
    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.

  • 83.
    Oglęcka, Kamila
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundberg, Pontus
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Magzoub, Mazin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, L. E. Göran
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Relevance of the N-terminal NLS-like sequence for membrane interactions of the Prion protein2007In: Biochimica et Biophysica Acta. MR. Reviews on Biomembranes, ISSN 0304-4157, E-ISSN 1879-257X, Vol. 1778, no 1, p. 206-213Article in journal (Refereed)
    Abstract [en]

    We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23–28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1–28 (mPrPp(1–28)) and 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.

  • 84.
    Oglęcka, Kamila
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundberg, Pontus
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Magzoub, Mazin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, L. E. Göran
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects2008In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1778, no 1, p. 206-213Article in journal (Refereed)
    Abstract [en]

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

  • 85.
    Papadopoulos, E
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oglecka, K
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, L
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jarvet, J
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wright, PE
    Dyson, HJ
    Gräslund, A
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    NMR solution structure of the peptide fragment 1-30, derived from unprocessed mouse Doppel protein, in DHPC micelles.2006In: Biochemistry, ISSN 0006-2960, Vol. 45, no 1, p. 159-66Article in journal (Refereed)
    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.

  • 86.
    Papadopoulos, Evangelos
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Collet, Jean-Francois
    Vukojevic, Vladana
    Billeter, Martin
    Holmgren, Arne
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vlamis-Gardikas, Alexios
    Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli2012In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1824, no 12, p. 1401-1408Article in journal (Refereed)
    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).

  • 87.
    Papapopoulos, Evangelos
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Billeter, Martin
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    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 motifs2007In: Biomol. NMR Assign., ISSN 1874-2718 (Print) 1874-270X (Online), Vol. 1, p. 217-219Article in journal (Refereed)
  • 88.
    Peralvarez-Marin, Alex
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mateos, Laura
    Zhang, Ce
    Singh, Shalini
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cedazo-Minguez, Angel
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Morozova-Roche, Ludmilla
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Influence of Residue 22 on the Folding, Aggregation Profile, and Toxicity of the Alzheimer's Amyloid beta Peptide2009In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 97, no 1, p. 277-285Article in journal (Refereed)
    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.

  • 89.
    Popović-Bijelić, Ana
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kowol, Christian R.
    Lind, Maria E. S.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Luo, Jinghui
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Himo, Fahmi
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Enyedy, Éva A.
    Arion, Vladimir B.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): A combined experimental and theoretical study2011In: European Journal of Inorganic Chemistry, ISSN 1434-1948, E-ISSN 1099-1948, Vol. 105, no 11, p. 1422-1431Article in journal (Refereed)
    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.

  • 90.
    Popović-Bijelić, Ana
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Voevodskaya, Nina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Domkin, Vladimir
    Thelander, Lars
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Metal binding and activity of ribonucleotide reductase protein R2 mutants: Conditions for formation of the mixed manganese-iron cofactor2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 27, p. 6532-6539Article in journal (Refereed)
    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.

  • 91. Richman, Michal
    et al.
    Wilk, Sarah
    Chemerovski, Marina
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wahlström, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rahimipour, Shai
    In Vitro and Mechanistic Studies of an Antiamyloidogenic Self-Assembled Cyclic D,L-alpha-Peptide Architecture2013In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 135, no 9, p. 3474-3484Article in journal (Refereed)
    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.

  • 92.
    Sahlin, Margareta
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Cho, Kyung-Bin
    Department of Biochemistry and Biophysics.
    Pötsch, Stephan
    Department of Biochemistry and Biophysics.
    Lytton, Simon D
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Huque, Yasmin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gunther, Michael R
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Mason, Ronald P
    Gräslund, Astrid
    Department of Biochemistry and Biophysics.
    Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli.2002In: J Biol Inorg Chem, ISSN 0949-8257, Vol. 7, no 1-2, p. 74-82Article in journal (Other academic)
    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.

  • 93. Shafaat, Hannah S.
    et al.
    Griese, Julia J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pantazis, Dimitrios A.
    Roos, Katarina
    Stockholm University, Faculty of Science, Department of Physics.
    Andersson, Charlotta S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Popovic-Bijelic, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Siegbahn, Per E. M.
    Stockholm University, Faculty of Science, Department of Physics.
    Neese, Frank
    Lubitz, Wolfgang
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cox, Nicholas
    Electronic Structural Flexibility of Heterobimetallic Mn/Fe Cofactors: R2lox and R2c Proteins2014In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, no 38, p. 13399-13409Article in journal (Refereed)
    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.

  • 94. Sigfridsson, Kajsa G. V.
    et al.
    Chernev, Petko
    Leidel, Nils
    Popovic-Bijelic, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haumann, Michael
    Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 14, p. 9648-9661Article in journal (Refereed)
    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.

  • 95. Tanaka, Gen
    et al.
    Nakase, Ikuhiko
    Fukuda, Yasunori
    Masuda, Ryo
    Oishi, Shinya
    Shimura, Kazuya
    Kawaguchi, Yoshimasa
    Takatani-Nakase, Tomoka
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Okawa, Katsuya
    Matsuoka, Masao
    Fujii, Nobutaka
    Hatanaka, Yasumaru
    Futaki, Shiroh
    CXCR4 Stimulates Macropinocytosis: Implications for Cellular Uptake of Arginine-Rich Cell-Penetrating Peptides and HIV2012In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 19, no 11, p. 1437-1446Article in journal (Refereed)
    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.

  • 96. Taqi, Malik Mumtaz
    et al.
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Yamskova, Olga
    Madani, Fatemeh
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bazov, Igor
    Luo, Jinghui
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zubarev, Roman
    Verbeek, Dineke
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bakalkin, Georgy
    Conformation Effects of CpG Methylation on Single-Stranded DNA Oligonucleotides: Analysis of the Opioid Peptide Dynorphin-Coding Sequences2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 6, p. e39605-Article in journal (Refereed)
    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.

  • 97.
    Teixeira, Pedro F.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Masuyer, Geoffrey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pinho, Catarina M.
    Branca, Rui M. M.
    Kmiec, Beata
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wallin, Cecilia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berntsson, Ronnie P. -A.
    Ankarcrona, Maria
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lehtiö, Janne
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 3, p. 348-362Article in journal (Refereed)
    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.

  • 98.
    Tiiman, Ann
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jarvet, Jüri
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. The National Institute of Chemical Physics and Biophysics, Estonia.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vukojevic, Vladana
    Heterogeneity and Turnover of Intermediates during Amyloid-beta (A beta) Peptide Aggregation Studied by Fluorescence Correlation Spectroscopy2015In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, no 49, p. 7203-7211Article in journal (Refereed)
    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.

  • 99.
    Tiiman, Ann
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Luo, Jinghui
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Oxford, UK.
    Wallin, Cecilia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Olsson, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindgren, Joel
    Jarvet, Jϋri
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. The National Institute of Chemical Physics and Biophysics, Estonia.
    Roose, Per
    Sholts, Sabrina B.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. National Museum of Natural History, USA.
    Rahimipour, Shai
    Abrahams, Jan Pieter
    Eriksson Karlström, Amelie
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Specific Binding of Cu(II) Ions to Amyloid-Beta Peptides Bound to Aggregation-Inhibiting Molecules or SDS Micelles Creates Complexes that Generate Radical Oxygen Species2016In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 54, no 3, p. 971-982Article in journal (Refereed)
    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.

  • 100.
    Tiklová, Katarína
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Senti, Kirsten-André
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Wang, Shenqiu
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Samakovlis, Christos
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila2010In: Nature Cell Biology, ISSN 1465-7392, E-ISSN 1476-4679, Vol. 12, no 11, p. 1071-1077Article in journal (Refereed)
    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.

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