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  • 1.
    Guterstam, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Madani, Fatemeh
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hirose, Hisaaki
    Takeuchi, Toshihide
    Futaki, Shiroh
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate2009In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, no 12, p. 2509-2517Article in journal (Refereed)
    Abstract [en]

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

  • 2.
    Madani, Fatemeh
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biophysical studies of peptides with functions in biotechnology and biology2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    My thesis concerns spectroscopic studies (NMR, CD and fluorescence) of peptides with functions in biotechnology and biology, and their interactions with a model membrane (large unilamellar phospholipid vesicles).

    The resorufin-based arsenical hairpin binder (ReAsH) bound to a short peptide is a useful fluorescent tag for genetic labeling of proteins in living cells. A hairpin structure with some resemblance to type II β-turn was determined by NMR structure calculations (Paper I).

    Cell-penetrating peptides (CPPs) are short (30-35 residues), often rich in basic amino acids such as Arg. They can pass through the cell membrane and deliver bioactive cargoes, making them useful for biotechnical and pharmacological applications. The mechanisms of cellular uptake and membrane translocation are under debate. Understanding the mechanistic aspects of CPPs is the major focus of Papers II, III, and IV.

    The effect of the pyrenebutyrate (PB) on the cellular uptake, membrane translocation and perturbation of several CPPs from different subgroups was investigated (Paper II). We concluded that both charge and hydrophobicity of the CPP affect the cellular uptake and membrane translocation efficiency.

    Endosomal escape is a crucial challenge for the CPP applications. We modeled the endosome and endosomal escape for different CPPs to investigate the corresponding molecular mechanisms (Papers III and IV). Hydrophobic CPPs were able to translocate across the model membrane in the presence of a pH gradient, produced by bacteriorhodopsin proton pumping, whereas a smaller effect was observed for hydrophilic CPPs.

    Dynorphin A (Dyn A) peptide mutations are associated with neurodegenerative disorders, without involvement of the opioid receptors. The non-opioid activities of Dyn A may involve membrane perturbations. Model membrane-perturbations by three Dyn A mutants were investigated (Paper V). The results showed effects to different degrees largely in accordance with their neurotoxic effects.

  • 3.
    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.

  • 4.
    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.

  • 5.
    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).

  • 6.
    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.

  • 7.
    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.

  • 8. 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.

  • 9.
    Unnerståle, Sofia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Madani, Fatemeh
    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-perturbing properties of two Arg-rich paddle domains from voltage-gated sensors in the KvAP and HsapBK K+ channels2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 19, p. 3982-3992Article in journal (Refereed)
    Abstract [en]

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

  • 10.
    Vasconcelos, Luís
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Madani, Fatemeh
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Arukuusk, Piret
    Pärnaste, Ly
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Effects of cargo molecules on membrane perturbation caused by transportan10 based cell-penetrating peptides2014In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1838, no 12, p. 3118-3129Article in journal (Refereed)
    Abstract [en]

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

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