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  • 201.
    Holm, Tina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pooga, Margus
    Lindgren, Maria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ulo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Studying the uptake of cell-penetrating peptides2006Inngår i: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 1, nr 2, s. 1001-1005Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.

  • 202.
    Holm, Tina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Räägel, Helin
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hein, Margot
    Mäe, Maarja
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pooga, Margus
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity2011Inngår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1808, nr 6, s. 1544-1551Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

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

  • 203.
    Holmlund, Linda
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cortes Toro, Veronica
    Iverfeldt, Kerstin
    Additive effects of amyloid β fragment and interleukin-1β on IL-6 secretion in rat primary glial cultures.2002Inngår i: International Journal of Molecular Medicine, ISSN 1107-3756, Vol. 10, s. 245-250Artikkel i tidsskrift (Fagfellevurdert)
  • 204. Holmström, Tim H.
    et al.
    Mialon, Antoine
    Kallio, Marko
    Nymalm, Yvonne
    Mannermaa, Leni
    Holm, Tina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Black, Elizabeth
    Gillespie, David
    Salminen, Tiina A.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Valdez, Benigno C.
    Westermarck, Jukka
    c-Jun supports ribosomal RNA processing and nucleolar localization of RNA helicase DDX212008Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, nr 11, s. 7046-7053Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The molecular mechanisms by which the AP-1 transcription factor c-Jun exerts its biological functions are not clearly understood. In addition to its well established role in transcriptional regulation of gene expression, several reports have suggested that c-Jun may also regulate cell behavior by non-transcriptional mechanisms. Here, we report that small interfering RNA-mediated depletion of c-Jun from mammalian cells results in inhibition of 28 S and 18 S rRNA accumulation. Moreover, we show that c-Jun depletion results in partial translocation of RNA helicase DDX21, implicated in rRNA processing, from the nucleolus to the nucleoplasm. We demonstrate that DDX21 translocation is rescued by exogenous c-Jun expression and that c-Jun depletion inhibits rRNA binding of DDX21. Furthermore, the direct interaction between c-Jun and DDX21 regulates nucleolar localization of DDX21. These results demonstrate that in addition to its transcriptional effects, c-Jun regulates rRNA processing and nucleolar compartmentalization of the rRNA processing protein DDX21. Thus, our results demonstrate a nucleolar mechanism through which c-Jun can regulate cell behavior. Moreover, these results suggest that the phenotypes observed previously in c-Jun-depleted mouse models and cell lines could be partly due to the effects of c-Jun on rRNA processing.

  • 205. Honaker, Matthew T.
    et al.
    Acchione, Mauro
    Zhang, Wei
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Atkins, William M.
    Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites2013Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 25, s. 18599-18611Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning induced fit versus conformational selection has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1-1 (GSTA1-1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1-1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that local molten globule behavior optimizes detoxication enzymes.

  • 206. Howl, John
    et al.
    Matou-Nasri, Sabine
    West, David C.
    Farquhar, Michelle
    Slaninova, Jirina
    Östenson, Claes-Göran
    Zorko, Matjaz
    Östlund, Pernilla
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kumar, Shant
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    McKeating, Jane
    Jones, Sarah
    Bioportide: an emergent concept of bioactive cell penetrating peptides2012Inngår i: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 69, nr 17, s. 2951-2966Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.

  • 207. Huenchuguala, Sandro
    et al.
    Munoz, Patricia
    Zavala, Patricio
    Villa, Monica
    Cuevas, Carlos
    Ahumada, Ulises
    Graumann, Rebecca
    Nore, Beston F.
    Couve, Eduardo
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Paris, Irmgard
    Segura-Aguilar, Juan
    Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction2014Inngår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 10, nr 4, s. 618-630Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit H-3-dopamine uptake, which is inhibited by 2 mu M of nomifensine and 15 mu M of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 mu M purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 mu M aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A(1), and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A(1) pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/-tubulin (tubulin, ) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.

  • 208. Hyvonen, Maija
    et al.
    Enback, Juulia
    Huhtala, Tuulia
    Lammi, Johanna
    Sihto, Harri
    Weisell, Janne
    Joensuu, Heikki
    Rosenthal-Aizman, Katri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Narvanen, Ale
    Bergers, Gabriele
    Laakkonen, Pirjo
    Novel Target for Peptide-Based Imaging and Treatment of Brain Tumors2014Inngår i: Molecular Cancer Therapeutics, ISSN 1535-7163, E-ISSN 1538-8514, Vol. 13, nr 4, s. 996-1007Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Malignant gliomas are associated with high mortality due to infiltrative growth, recurrence, and malignant progression. Even with the most efficient therapy combinations, median survival of the glioblastoma multiforme (grade 4) patients is less than 15 months. Therefore, new treatment approaches are urgently needed. We describe here identification of a novel homing peptide that recognizes tumor vessels and invasive tumor satellites in glioblastomas. We demonstrate successful brain tumor imaging using radiolabeled peptide in whole-body SPECT/CT imaging. Peptide-targeted delivery of chemotherapeutics prolonged the lifespan of mice bearing invasive brain tumors and significantly reduced the number of tumor satellites compared with the free drug. Moreover, we identified mammary-derived growth inhibitor (MDGI/H-FABP/FABP3) as the interacting partner for our peptide on brain tumor tissue. MDGI was expressed in human brain tumor specimens in a grade-dependent manner and its expression positively correlated with the histologic grade of the tumor, suggesting MDGI as a novel marker for malignant gliomas. Mol Cancer Ther; 13(4); 996-1007. (C)2014 AACR.

  • 209.
    Hällbrink, Mattias
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Karelson, Mati
    Prediction of Cell-Penetrating Peptides2015Inngår i: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, Springer-Verlag New York, 2015, s. 39-58Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    The in silico methods for the prediction of the cell-penetrating peptides are reviewed. Those include the multivariate statistical methods, machine-learning methods such as the artificial neural networks and support vector machines, and molecular modeling techniques including molecular docking and molecular dynamics.

    The applicability of the methods is demonstrated on the basis of the exemplary cases from the literature.

  • 210.
    Hällbrink, Mattias
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Prediction of cell-penetrating peptides2007Inngår i: Handbook of cell-penetrating peptides / [ed] Ülo Langel, Boca Raton: CRC Press, 2007, 2, s. 77-85Kapittel i bok, del av antologi (Fagfellevurdert)
  • 211.
    Hälldin, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Oxidative stress and alterations in the mammalian iron metabolism: A study on iron, inflammation, oxidative stress and neurodegeneration2007Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Iron is essential for all living organisms, from bacteria to man for a broad variety of biological functions, including oxygen transport and DNA synthesis. However, iron can also be toxic, due to its involvement in the formation of highly reactive oxygen species through Fenton chemistry. Iron has been suggested to be involved in the pathology of several neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease as well as prion diseases, such as Creutzfeldt-Jakob’s disease and scrapie. The work presented in this thesis is linked to the intracellular iron metabolism, oxidative stress and inflammation.

    Studies of iron metabolism in scrapie infected neuroblastoma N2a cells indicate that the scrapie infection is lowering the levels of both total iron and the labile iron pool. We also detected variations in the activities of iron regulatory protein 1 and 2 in the scrapie infected cells as compared to control cells. In addition, we have used DNA microarrays to compare the expression levels of mRNAs corresponding to several genes important for the antioxidative defense such as glutathione peroxidase and glutathione transferases between scrapie infected N2a and control cells. Our results suggest that the scrapie infected cells show differences in the expression of important genes involved in the cellular defense against oxidative stress as compared to control cells.

    We have also studied alterations in the iron metabolism as a response to the bacterial toxin lipopolysaccharide (LPS). LPS treatment leads to a decrease of transferrin receptor protein (TfR) and to an increase of ferritin mRNA levels in N2a and BV-2 cells. The decrease of TfR expression is dependent on reactive oxygen species (ROS) and nitric oxide (NO) species, whereas the increase of ferritin mRNA is independent of both ROS and NO formation.

    Finally, we have studied the effect of sulfide on iron regulation in RD4 cells. We detected a substantial sulfide-induced increase in the labile iron pool. We also measured markedly elevated levels of ferritin protein and a decrease in IRP2 protein, indicating that the released iron was made bioavailable.

  • 212.
    Hälldin, Jonas
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kask, Indrek
    Metspalu, Andres
    Land, Tiit
    Microarray analysis of gene expression in scrapie-infected neuroblastoma cells: Implication of oxidative stressManuskript (Annet vitenskapelig)
  • 213.
    Hälldin, Jonas
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Land, Tiit
    Sulfide increases labile iron pool in RD4 cells2008Inngår i: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 21, nr 2, s. 127-131Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A linkage between sulfur and iron metabolism has been suggested since sulfide has the ability to release iron from ferritin in the presence of iron acceptors in vitro. Nevertheless, this linkage is still lacking evidence in vivo as well as in cellular models. In this study we have treated human RD4 skeletal muscle cells with sodium sulfide and measured the level of the labile iron pool (LIP) as well as the intracellular sulfide concentration. We have also detected the amounts of L-ferritin protein as well as the iron regulatory protein 2 (IRP2). The sulfide treatment resulted in a 100% increase in the amount of LIP after 1 and 2 h. We also found that the raise of the LIP levels was coupled to an elevation of the amounts of intracellular sulfide that increased by 60%. The bioavailability of the released iron was confirmed by a 100% increase in L-ferritin protein as well as a 60% decrease of the IRP2 protein levels. These results suggest that there is a linkage between sulfur metabolism and intracellular iron regulation in mammalian cells.

  • 214. Ito, Mika
    et al.
    Shibata, Aya
    Zhang, Jie
    Hiroshima, Michio
    Sako, Yasushi
    Nakano, Yukiko
    Kojima-Aikawa, Kyoko
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Shuto, Satoshi
    Ito, Yoshihiro
    Morgenstern, Ralf
    Abe, Hiroshi
    Universal Caging Group for the in-Cell Detection of Glutathione Transferase Applied to 19F NMR and Bioluminogenic Probes2012Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, nr 10, s. 1428-1432Artikkel i tidsskrift (Fagfellevurdert)
  • 215.
    Ivanova, Elena V.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Caspase activation in human neuroblastoma cells: mechanisms and spatiotemporal aspects2015Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Apoptosis is one of the modes of programmed cell death, in which several members of the caspase family of proteases play the central role. However, activation of apoptotic caspases does not necessarily lead to cell death. Instead, these caspases may mediate, for instance, differentiation or synaptic plasticity, if their activity is restricted in space and time. Such localized caspase activation has been also implicated in the initial stages of neurodegeneration. In order to assess this kind of events at a subcellular level, our research group has previously constructed tau-anchored FRET-based caspase sensors (tAFSs). Here, we demonstrate that localization of tAFSs to the cytoskeleton results in enrichment of the sensors in neuritic processes and enables increased spatiotemporal resolution for live cell imaging of caspase activation, as compared to soluble FRET sensors. This feature is particularly beneficial for investigation of neurodegeneration-related processes.

    tAFSs were further employed for investigation of caspase activation in neuroblastoma, an extracranial solid pediatric tumor. Tumor necrosis factor-related apoptosis inducing factor (TRAIL) is a promising candidate for cancer treatment due to its ability to selectively trigger apoptosis malignant cells. However, many cancer cells, including neuroblastoma, acquire resistance to TRAIL. Here, we show that in S-type neuroblastoma cell lines, TRAIL resistance is dependent on incomplete activation of apoptotic caspase-3. Sensitization to TRAIL was achieved with protein kinase C (PKC)-inhibiting compounds, suggesting a role for this kinase in blocking the apoptotic response to TRAIL. This effect of PKC could possibly involve stabilization of XIAP, an endogenous caspase inhibitor, as PKC inhibition, in combination with TRAIL treatment, led to downregulation of XIAP.

  • 216.
    Ivanova, Elena V.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gatsinzi, Tom
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Anchoring of FRET Sensors-A Requirement for Spatiotemporal Resolution2016Inngår i: Sensors, ISSN 1424-8220, E-ISSN 1424-8220, Vol. 16, nr 5, artikkel-id 703Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling.

  • 217.
    Ivanova, Elena V.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gatsinzi, Tom
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Increased spatiotemporal resolution of caspase activation by anchoring FRET-based sensors to cytoskeletonManuskript (preprint) (Annet vitenskapelig)
  • 218.
    Jacobsen, Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Processing of the APP family by the α-secretases ADAM10 and TACE2010Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Alzheimer’s disease (AD) is a progressive neurodegenerative disease, which is characterized by formation of amyloid plaques in the brain. The major constituent of these plaques is the hydrophobic peptide Aβ. Aβ accumulation is considered to be the main cause of the pathology seen in AD brains. Aβ is produced through sequential cleavage of the amyloid precursor protein (APP). APP can be processed by two different enzymatic pathways. Formation of Aβ requires cleavage of APP by β- and γ-secretase. However, most proteolytic processing of APP does not result in Aβ formation. Instead, APP is mainly cleaved by α-secretase, which not only precludes formation of the toxic Aβ peptide but also generates the neuroprotective sAPPα fragment. Increasing the α-secretase processing of APP is thereby a potential therapeutic strategy for AD. APP is a member of a conserved gene family, also including the APP-like proteins-1 and -2 (APLP1 and APLP2). The APP family members have essential and overlapping functions and have been reported to be processed in a similar way by the same enzymes. The processing of all APP family members is increased in response to several stimuli, including retinoic acid (RA) and insulin-like growth factor-1 (IGF-1), which also induce a shift towards α-secretase processing. The aim of this thesis was to investigate the mechanisms and signaling involved in induced α-secretase processing of the APP family. The main α-secretase candidates are ADAM10 and TACE. In this thesis we wanted to study the effects on expression levels of ADAM10 and TACE during RA treatment. We also wanted to investigate the mechanism behind IGF-1-induced processing of APP and APLP2. We found that both ADAM10 and TACE are up-regulated in response to RA, but that the signaling pathways involved differed between the two enzymes. Similarly, we showed that IGF-1-induced processing of APLP2, but not of APP, is dependent on PKC. Furthermore, we showed that ADAM10 is the main α-secretase for APP, whereas TACE cleaves APLP2 in response to IGF-1. We conclude that although APP and APLP2 proteolytic processing are induced by the same stimuli, the processing is dependent on different signaling pathways and processing enzymes, which in turn are differentially regulated.

  • 219.
    Jacobsen, Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    α-Secretase processing of the Alzheimer amyloid-β precursor protein and its homolog APLP22013Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The amyloid-β precursor protein (APP) has been widely studied due to its role in Alzheimer´s disease (AD). When APP is sequentially cleaved by β- and γ-secretase, amyloid-β (Aβ) is formed. Aβ is prone to aggregate and is toxic to neurons. However, the main processing pathway for APP involves initial cleavage at the α-site, within the Aβ region, instead generating a neuroprotective soluble fragment, sAPPα. APP is a member of a protein family, also including the proteins APLP1 and APLP2, which are processed in a similar way as APP. In addition, K/O studies in mice have shown that the three proteins have overlapping functions where APLP2 play a key physiological role. The aim of this thesis was to study mechanisms underlying the α-secretase processing of APP and APLP2. We have used the human neuroblastoma cell-line SH-SY5Y as a model system and stimulated α-secretase processing with insulin-like growth factor-1 (IGF-1) or retinoic acid (RA). Our results show that the stimulated α-site cleavage of APP and APLP2 is regulated by different signaling pathways and that the cleavage is mediated by different enzymes. APP was shown to be cleaved by ADAM10 in a PI3K-dependent manner, whereas APLP2 was cleaved by TACE in a PKC-dependent manner. We further show that protein levels and maturation of ADAM10 and TACE is increased in response to RA, mediated by a PI3K- or PKC-dependent signaling pathway, respectively. Another focus of our research has been O-GlcNAcylation, a dynamic post-translational modification regulated by the enzymes O-GlcNAc transferase and O-GlcNAcase (OGA). We show that decreased OGA activity stimulates sAPPα secretion, without affecting APLP2 processing. We further show that ADAM10 is O-GlcNAcylated. Lastly, we show that APP can be manipulated to be cleaved in a similar way as APLP2 during IGF-1 stimulation by substituting the E1 domain in APP with the E1 domain in APLP2. Together our results show distinct α-site processing mechanisms of APP and APLP2.

  • 220.
    Jacobsen, Kristin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Adlerz, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Multhaup, Gerd
    Institute for chemistry and biochemistry, Free University of Berlin.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Insulin-like growth factor-1 (IGF-1)-induced processing of amyloid-β precursor protein (APP) and APP-like protein 2 is mediated by different metalloproteinases2010Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, nr 14, s. 10223-10231Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    α-Secretase cleavage of the amyloid precursor protein (APP) is of great interest since it prevents the formation of the Alzheimer-linked amyloid-β peptide. APP belongs to a conserved gene family including the two paralogues APP-like protein (APLP) 1 and 2. Insulin-like growth factor-1 (IGF-1) stimulates the shedding of all three proteins. IGF-1-induced shedding of both APP and APLP1 is dependent on phosphatidylinositol 3-kinase (PI3-K), whereas sAPLP2 secretion is independent of this signaling pathway. Here, we used human neuroblastoma SH-SY5Y cells to investigate the involvement of protein kinase C (PKC) in the proteolytic processing of endogenously expressed members of the APP family. Processing was induced by IGF-1 or retinoic acid, another known stimulator of APP a-secretase shedding. Our results show that stimulation of APP and APLP1 processing involves multiple signaling pathways, whereas APLP2 processing is mainly dependent on PKC. Next, we wanted to investigate if the difference in the regulation of APLP2 shedding compared to APP shedding could be due to involvement of different processing enzymes. We focused on the two major a-secretase candidates ADAM10 and TACE, which both are members of the ADAM (a disintegrin and metalloprotease) family. Shedding was analyzed in the presence of the ADAM10 inhibitor GI254023X, or after transfection with siRNA targeted against TACE. The results clearly demonstrate that different α-secretases are involved in IGF-1-induced processing. APP is mainly cleaved by ADAM10, whereas APLP2 processing is mediated by TACE. Finally, we also show that IGF-1 induces PKC-dependent phosphorylation of TACE.

  • 221.
    Jacobsen, Kristin T
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Amyloid precursor protein and its homologues: a family of proteolysis-dependent receptors.2009Inngår i: Cellular and molecular life sciences : CMLS, ISSN 1420-9071, Vol. 66, nr 14, s. 2299-2318Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The Alzheimer's amyloid precursor protein (APP) belongs to a conserved gene family that also includes the mammalian APLP1 and APLP2, the Drosophila APPL, and the C. elegans APL-1. The biological function of APP is still not fully clear. However, it is known that the APP family proteins have redundant and partly overlapping functions, which demonstrates the importance of studying all APP family members to gain a more complete picture. When APP was first cloned, it was speculated that it could function as a receptor. This theory has been further substantiated by studies showing that APP and its homologues bind both extracellular ligands and intracellular adaptor proteins. The APP family proteins undergo regulated intramembrane proteolysis (RIP), generating secreted and cytoplasmic fragments that have been ascribed different functions. In this review, we will discuss the APP family with focus on biological functions, binding partners, and regulated processing.

  • 222.
    Jacobsen, Kristin T.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    O-GlcNAcylation increases non-amyloidogenic processing of the amyloid-beta precursor protein (APP)2011Inngår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 404, nr 3, s. 882-886Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The amyloid-beta precursor protein (APP) was shown to be O-GlcNAcylated 15 years ago, but the effect of this modification on APP processing and formation of the Alzheimer's disease associated amyloid-beta (A beta) peptide has so far not been investigated. Here, we demonstrate with pharmacological tools or siRNA that O-GlcNAcase and O-GlcNAc transferase regulate the level of O-GlcNAcylated APP. We also show that O-GlcNAcylation increases non-amyloidogenic alpha-secretase processing, resulting in increased levels of the neuroprotective sAPP alpha fragment and decreased A beta secretion. Our results implicate O-GlcNAcylation as a potential therapeutic target for Alzheimer's disease.

  • 223.
    Jacobsen, Kristin T.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The E1 domain of APP and APLP2 determines α-secretase specificityManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    The α-secretase cleavage of the amyloid-β precursor protein (APP) precludes the formation of amyloid-β (Aβ), the main constituent of senile plaques in Alzheimer´s disease (AD). Stimulation of α-secretase processing may thereby constitute an important therapeutical strategy. APP belongs to a conserved protein family including the APP-like protein 2 (APLP2). Although the proteins are sequentially processed in a similar way, we have previously shown that insulin-like factor-1 (IGF-1) - and retinoic acid (RA)-induced α-secretase processing of APP and APLP2 is mediated by different enzymes. APP was shown to be cleaved by the α-secretase ADAM10 in a PI3K-dependent manner, whereas APLP2 was cleaved by the α-secretase TACE in a PKC-dependent manner. To better understand the mechanism underlying this difference in α-secretase processing between these two homologous proteins, we aimed to determine which part of APP that was essential for making it a better substrate for ADAM10 than for TACE during stimulated conditions. We constructed a chimeric protein, were the E1 domain of APP was substituted with the corresponding domain of APLP2. Our results demonstrate that the APP/E1/APLP2 chimer is successfully expressed and secreted into the cell medium of transiently transfected SH-SY5Y cells. Pharmacological inhibition demonstrate that the IGF-1-induced processing of the chimer is PI3K-independent but dependent on PKC, as previously shown for the APLP2 processing. Our result indicates that the E1 domain in APP determines its specificity towards ADAM10 over TACE.

  • 224.
    Jacobsen, Kristin T.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Strååt, Ylva
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Koistinen, Niina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    O-GlcNAcylation of the α-secretase ADAM10 selectively affects APP processing in neuron-like cellsManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    α-Secretase processing of APP has recently gained more interest, highlighting its potential as a therapeutic target to prevent Alzheimer’s disease (AD). We have previously shown that O-GlcNAcylation stimulates α-secretase processing of APP, concomitantly with decreased Aβ secretion. O-GlcNAcylation has previously been linked to AD since the levels of O-GlcNAcylated proteins are decreased in AD brains. Here, we have further investigated the mechanism behind α-secretase processing of APP in response to increased O-GlcNAcylation. Our results shown that APP is not O-GlcNAcylated during the conditions used in this study. Instead, we demonstrate that the α-secretase ADAM10 is O-GlcNAcylated and that APP cell surface localization is enhanced in response to increased O-GlcNAcylation. Furthermore, the effects of O-GlcNAcylation on APP processing are cell-type specific, only affecting sAPPα secretion in neuroblastoma cell-lines.

  • 225.
    Jaffer Ali, Mohammed Hakim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Multifaceted roles of the transmembrane nuclear envelope protein, Samp12017Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The eukaryotic nuclear envelope (NE), separates the nucleoplasm from cytoplasm and is made up of two concentric lipid membranes, the outer and the inner nuclear membranes (ONM and INM), the nuclear pore complexes (NPCs) and an underlying filamentous nuclear lamina. The INM contains hundreds of unique transmembrane proteins of which only a handful have been characterized. In this thesis, I aimed to understand the functional organization of proteins in the nuclear envelope and I focused on investigating the functions of a recently identified INM transmembrane protein, Samp1. We have developed a novel and robust approach, MCLIP, to identify specific protein-protein interactions taking place in live cells. Using MCLIP, we have shown that Samp1 interacts with proteins of the LINC complex, the nuclear lamina and components of the mitotic spindle. Samp1's specific interactions with a variety of binding partners, suggest that Samp1 plays important roles both in interphase and in mitosis.  We have also shown that Samp1 can provide a binding site at the INM for the GTPase Ran, a master regulator of protein interactions in interphase and in mitosis. Furthermore, we have also investigated the role of Samp1 in cell differentiation using two independent model systems. In human iPSCs, ectopic expression of Samp1 promoted differentiation despite pluripotent culture conditions. In C2C12 myoblast, depletion of Samp1 completely blocked differentiation into myotubes. The two studies complement each other and suggest that Samp1 has a strong differentiation promoting activity. Taken together, the findings in this thesis, give insights on the unexpected and unforeseen roles played by a transmembrane protein in different fundamental cellular process.

  • 226.
    Jaffer Ali, Mohammed Hakim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Nuclear envelope protein interaction studies2014Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The nuclear envelope (NE) separating the nucleoplasm from cytoplasm consists of two concentric lipid membranes, the outer (ONM) and inner (INM) nuclear membranes, the nuclear pore complexes (NPCs) and an underlying nuclear lamina network. The INM contains more than 100 unique transmembrane proteins of which only a few have been characterized. This thesis is focused on one of these INM proteins, Samp1 (Spindle associated membrane protein 1)

    Protein-protein interactions in the NE have been difficult to study due to the resistance of NE proteins to extraction. We have established a reversible in vivo crosslinking immunoprecipitation method called, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation) to overcome this problem. Using MCLIP we were able to show that, Samp1 specifically interacts with Emerin, Lamin B1, Sun1 and the small GTPase Ran. We also showed that, the nucleoplasmic domain of Samp1 and Emerin can interact with each other directly.

    Furthermore, we investigated the functional role of Samp1 in mitosis. Samp1 depletion gave rise to aneuploid phenotypes and signs of destabilization of the mitotic spindle. Using MCLIP, in mitotic cells, we showed that, Samp1 interacts with Ran and Importin-β, two key players of mitotic spindle assembly. We observed that, Samp1 modulates the level of Importin-β and NuMA in the mitotic spindle, which may explain the mitotic defects and aberrant phenotypes observed in Samp1 depleted cells. These findings show that Samp1 plays an important role in spindle stabilization and chromosome segregation. 

  • 227.
    Jafferali, Mohammed Hakim
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    MCLIP Detection of Novel Protein-Protein Interactions at the Nuclear Envelope2016Inngår i: Intermediate Filament Associated Proteins / [ed] Katherine L. Wilson, Arnoud Sonnenberg, SAN DIEGO: ELSEVIER ACADEMIC PRESS INC , 2016, Vol. 569, s. 503-515Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    The organization and function of the nuclear envelope (NE) involves hundreds of nuclear membrane proteins and myriad protein-protein interactions, most of which are still uncharacterized. Many NE proteins interact stably or dynamically with the nuclear lamina or chromosomes. This can make them difficult to extract under non-denaturing conditions, and greatly limits our ability to explore and identify functional protein interactions at the NE. This knowledge is needed to understand nuclear envelope structure and the mechanisms of human laminopathy diseases. This chapter provides detailed protocols for MCLIP (membrane cross-linking immunoprecipitation) identification of novel protein-protein interactions in mammalian cells.

  • 228.
    Jafferali, Mohammed Hakim
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hasan, Mehedi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Spindle associated membrane protein 1 (Samp1) is required for the differentiation of muscle cells2017Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikkel-id 16655Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Muscles are developed and regenerated in a differentiation process called myogenesis, which involves components of the nuclear envelope. We have investigated Samp1 (Spindle Associated Membrane Protein 1), a transmembrane nuclear envelope protein, which interacts with emerin and lamin A, both of which are linked to Emery-Dreifuss muscular dystrophy (EDMD). We found that the levels of Samp1 increased seven-fold during differentiation of mouse C2C12 muscle progenitor cells. To test if Samp1 could have a role in myogenesis we developed stable C2C12 knockdown cell lines expressing short hairpin RNA targeting Samp1 expression. The Samp1 depleted C2C12 cells displayed normal mobility and normal distribution of emerin and lamin A. However, Samp1 depletion increased ERK signaling and completely blocked differentiation of C2C12 cells, which failed to express myogenic marker proteins and failed to form myotubes. The block in myogenesis in Samp1 depleted cells was completely rescued by ectopic expression of RNAi resistant human Samp1, showing that Samp1 is required for muscle differentiation.

  • 229.
    Jafferali, Mohammed Hakim
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Vijayaraghavan, Balaje
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Crafoord, Ellinor
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gudise, Santhosh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Karolinska Institutet, Sweden.
    Larsson, Veronica J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells2014Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, nr 10, s. 2399-2403Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Investigating interactions of proteins in the nuclear envelope (NE) using co-immunoprecipitation (Co-IP) has previously been difficult or even impossible due to their inherent resistance to extraction. We have developed a novel method, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation), which takes advantage of a cell permeable crosslinker to enable effective detection and analysis of specific interactions of NE proteins in live cells using Western blot. Using MCLIP we show that, in U2OS cells, the integral inner nuclear membrane protein Samp1 interacts with Lamin B1, the LINC (Linker of nucleoskeleton and cytoskeleton) complex protein, Sun1 and the soluble small GTPase Ran. The results show that the previously detected in vitro interaction between Samp1 and Emerin also takes place in live cells. In vitro pull down experiments show, that the nucleoplasmic domains of Samp1 and Emerin can bind directly to each other. We also, show that MCLIP is suitable to coprecipitate protein interactions in different stages of the cell cycle.

  • 230.
    Jarsjö, Jerker
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för naturgeografi och kvartärgeologi (INK).
    Shibuo, Yoshihiro
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för naturgeografi och kvartärgeologi (INK).
    Destouni, Georgia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Spatial distribution of unmonitored inland water discharges to the sea2007Inngår i: Eos Trans. (2007) AGU, 88(52), Fall Meet. Suppl.: Abstract H43E-1675, 2007Konferansepaper (Annet vitenskapelig)
    Abstract [en]

    Eos Trans. AGU, 88(52), Fall Meet. Suppl., Abstract H43E-1675, 2007

  • 231.
    Jeppsson, Fredrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Characterization of Diagnostic Tools and Potential Treatments for Alzheimer’s Disease: PET ligands and BACE1 inhibitors2016Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Alzheimer’s disease (AD) is a very complex disorder and the most common form of dementia. The two pathological hallmarks of AD are extracellular amyloid-β (Aβ) plaques in cerebral cortex, and intraneuronal neurofibrillary tangles. In the early stages of the disease it can be difficult to accurately diagnose AD, as it is difficult to distinguish from normal signs of aging. There is thus a need for sensitive non-invasive tools, able to detect pathophysiological biomarker changes. One such approach is molecular imaging of Aβ plaque load in brain, using PET (positron emission tomography) ligands.

    We have developed and characterized two novel Aβ plaque neuroimaging PET ligands, AZD2184 and AZD4694. The 2-pyridylbenzothiazole derivate AZD2184, is a 11C-labeled PET ligand with a higher signal-to-background ratio compared to the widely used PET ligand PIB, a 11C-labeled phenylbenzothiazole based tool. This makes it possible to detect smaller changes in Aβ plaque deposition load, and therefore theoretically, also earlier diagnosis. A drawback with 11C-labeled PET ligands is the relatively short half-life. To meet the need for PET ligands with a longer half-life, we developed the pyridylbenzofuran derivate [18F]AZD4694. Although development of fluorinated radioligands is challenging due to the lipophilic nature of aromatic fluorine, we successfully developed a 18F-labeled PET ligand with a signal-to-background ratio matching PIB, the most widely used 11C-labeled PET ligand in clinical use. 3H-labeled derivates of AZD2184, AZD4694, and PIB, showed lower binding specificity towards Aβ plaques containing ApoE. The ApoE genotype per se did not significantly affect ligand binding, instead, the amount of ApoE incorporated to the Aβ plaques appears to be of importance for the binding characteristics of these amyloid PET ligands.

    Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid precursor protein (APP) to Aβ peptides, making BACE1 inhibition an attractive therapeutic target in AD. We developed and characterized three novel BACE1 inhibitors, AZD3839, AZ-4217, and AZD3293. AZD3839 and AZ-4217 contains an amidine group which interacts with the catalytic aspartases Asp-32 and Asp-228 of BACE1, effectively inhibiting the enzyme. All three compounds are potent and selective inhibitors of human BACE1, with in vitro potency demonstrated in several cellular models, including primary cortical neurons. All three compound exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aβ levels in several species, and two of the compounds (AZD3839 and AZD3293) were progressed into clinical trials.

  • 232. Jevtusevskaja, Jekaterina
    et al.
    Krolov, Katrin
    Tulp, Indrek
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification2017Inngår i: Expert Review of Molecular Diagnostics, ISSN 1473-7159, E-ISSN 1744-8352, Vol. 17, nr 4, s. 403-410Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Background: The use of rapid amplification methods to detect pathogens in biological samples is mainly limited by the amount of pathogens present in the sample and the presence of inhibiting substances. Inhibitors can affect the amplification efficiency by either binding to the polymerase, interacting with the DNA, or interacting with the polymerase during primer extension. Amplification is performed using DNA polymerase enzymes and even small changes in their activity can influence the sensitivity and robustness of molecular assaysMethods: The main purpose of this research was to examine which compounds present in urine inhibit polymerases with strand displacement activity. To quantify the inhibition, we employed quantitative loop-mediated isothermal amplificationResults: The authors found that the presence of BSA, Mg 2+, and urea at physiologically relevant concentrations, as well as acidic or alkaline conditions did not affect the activity of any of the tested polymerases. However, addition of salt significantly affected the activity of the tested polymerases.Conclusion: These findings may aid in the development of more sensitive, robust, cost effective isothermal amplification based molecular assays suitable for both point-of-care testing and on-site screening of pathogens directly from unprocessed urine which avoid the need for long and tedious DNA purification steps prior to amplification.

  • 233. Jevtusevskaja, Jekaterina
    et al.
    Uusna, Julia
    Andresen, Liis
    Krolov, Katrin
    Laanpere, Made
    Grellier, Tiia
    Tulp, Indrek
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Combination with antimicrobial peptide lyses improves loop-mediated isothermal amplification based method for Chlamydia trachomatis detection directly in urine sample2016Inngår i: BMC Infectious Diseases, ISSN 1471-2334, E-ISSN 1471-2334, Vol. 16, artikkel-id 329Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Chlamydia trachomatis is an obligate intracellular human pathogen and is the most common cause of sexually transmitted diseases affecting both men and women. The pathogen can cause prostatitis and epididymitis in men. In women, cervicitis, pelvic inflammatory disease, ectopic pregnancy and acute or chronic pelvic pain are frequent complications. More than half of C. trachomatis-positive patients have minimal or no symptoms, providing an ongoing reservoir for the infection. The lack of sensitive large-scale applicable point-of-care (POC) tests for C. trachomatis detection makes it difficult to diagnose chlamydia infection efficiently in resource-limited environments. Methods: A rapid and sensitive assay based on loop-mediated isothermal amplification method (LAMP) was combined with antimicrobial peptide lysis, which is able to detect at least 7 C. trachomatis pathogens per reaction directly from urine samples. Results: Our study comprising 91 first-void urine samples showed that specificity of the assay is 100 % and sensitivity 73 % when using antimicrobial peptide lysis mix. Additionally we demonstrate that our assay does not give any cross-reactivity with 30 pathogen's DNA potentially present in the urine samples. Furthermore, the assay's novel approach does not require purification or extraction of DNA from clinical sample prior to amplification, so the need for specialized equipment is eliminated. Conclusions: The whole procedure is significantly less laborious, less time-consuming and consequently less expensive for early detection and identification of infectious disease. C. trachomatis specific LAMP assay is relatively simple to perform and could therefore be applied in numerous POC settings.

  • 234.
    Jiang, Yang
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Soomets, Ursel
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Design and synthesis of cell-penetrating peptides2007Inngår i: Handbook of cell-penetrating peptides / [ed] Ülo Langel, Boca Raton: CRC Press, 2007, 2, s. 537-551Kapittel i bok, del av antologi (Fagfellevurdert)
  • 235. Jimenez-Andrade, Juan Miguel
    et al.
    Lundström, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sollenberg, Ulla E.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Castañeda-Hernandez, Gilberto
    Carlton, Susan M.
    Activation of peripheral galanin receptors: differential effects on nociception2006Inngår i: Pharmacology, Biochemistry and Behavior, ISSN 0091-3057, E-ISSN 1873-5177, Vol. 85, nr 1, s. 273-280Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Numerous reports suggest a significant role of peripheral galanin (GAL) in pain transmission; however, due to the lack of selective galanin receptor agonists and antagonists, the role of GAL receptors (GalR1-3) in pain transmission remains unclear. In this study, a new agonist, M617, that preferentially binds to GalR1, a GalR2 agonist (AR-M1896), and a GalR2 antagonist (M871) were tested in the periphery to elucidate the role of peripheral GalR1 and GalR2 in nociception. Ipsilateral, but not contralateral, hindpaw injection of M617 reduced capsaicin (CAP)-induced flinching by approximately 50%, suggesting that GalR1 activation produces anti-nociception. This anti-nociceptive effect was blocked by intraplantar injection of the non-selective GalR antagonist M35. In contrast ipsilateral, but not contralateral, intraplantar injection of GalR2 agonist AR-M1896 enhanced the CAP-induced nociception (1.7-fold). The GalR2 antagonist M871 blocked the pro-nociceptive effect of AR-M1896 in a dose-dependent manner. This antagonist had no effect on nociceptive behaviors induced by CAP alone. The data demonstrate that activation of peripheral GalR1 results in anti-nociception but activation of peripheral GalR2 produces pro-nociception. Thus, the use of these pharmacological tools may help to elucidate the contribution of GalR subtypes in nociceptive processing, identifying potential drug targets for the treatment of peripheral pain.

  • 236.
    Johansson, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides in protein mimicry and oligonucleotide delivery: Applications and mechanisms2008Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The plasma membrane functions as a barrier, restricting entry of hydrophilic pharmaceutical agents. Cell-penetrating peptides (CPPs) are capable of transporting bioactive cargos into the cell and have consequently been extensively investigated for their mechanism of entry and capability to deliver various cargos spanning from peptides to plasmids.

    The main aim of this thesis was to investigate the mechanism and capability of some of these CPPs to deliver mainly oligonucleotides and peptides into the cell. Oligonucleotides in the form of ds DNA decoy for sequestering of transcription factors or PNAs for redirection of splicing. In addition, peptides derived from the interaction interface of a tumor suppressor protein were investigated for their potential to combine a biological effect with internalization.

    Peptides with or without any cargo were predominantly dependent on some form of endocytic mechanism for internalization, substantiated by using a functional assay, where all tested CPPs were associated with endocytosis for delivery of splice correcting PNAs. A new CPP, M918 proved most efficient in promoting splice correction and internalized mainly via macropinocytosis. In addition, TP10 efficiently delivered dsDNA decoy oligonucleotides for sequestering of the transcription factor Myc with a concomitant biological response, i.e. reduced proliferation. Finally, for the first time, to our knowledge, a novel pro-apoptotic peptide with cell-penetrating properties was designed from the tumor suppressor p14ARF, which decreased proliferation and induced apoptosis in cancer cell-lines, potentially mimicking the full-length protein. Altogether, this thesis highlights the functionality of CPPs and the possibility to develop new CPPs with improved or new properties, having the potential to advance delivery of therapeutic compounds.

  • 237.
    Johansson, Henrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    El-Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Holm, Tina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mäe, Maarja
    Jaak, Jänes
    Maimets, Toivo
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein2008Inngår i: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 16, nr 1, s. 115-123Artikkel i tidsskrift (Fagfellevurdert)
  • 238. Johansson, Henrik J.
    et al.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Tartu University, Estonia.
    Mimicry of protein function with cell-penetrating peptides2011Inngår i: Cell-penetrating peptides: Methods and Protocols / [ed] Ülo Langel, Humana Press, 2011, s. 233-247Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    Proteins are essential components of cellular processes inside cells, and their interactions between each other and with genes are important for the normal physiological functioning of cells as well as for disease states. Modulating protein interactions by different means can potentially control these interactions and restore normal function to diseased cells. The ways to do so are multiple, and such efforts often begin with knowledge of potential target proteins in order to devise mediators that retain the function of the original protein, i.e., mimic the protein functions. An alternative strategy is to utilize protein mimics to inhibit target proteins rather than restoring the activity of a protein. The vast majority of protein ­mimics exploited to date have been designed to inhibit the activity of oncogenes or activate tumor suppressors for the purpose of tumor therapy. These protein mimics are usually based on small organic compounds or peptides, derived from interaction surfaces of the proteins, and in some cases, full proteins have been exploited. Although peptides and proteins are naturally highly specific and efficient inside cells, they suffer from low bioavailability resulting from their inability to enter cells. One strategy increasingly employed to facilitate the internalization of peptides and proteins has been to chemically conjugate them to cell-penetrating peptides (CPP) or to recombinantly express protein–CPP fusion constructs.

    This chapter provides an overview of some of the aspects of perturbing and mimicking protein interactions using peptides and proteins and CPP as transport vectors.

  • 239.
    Johansson, H.J.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL-Andaloussi, S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ü.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mimicking p14ARF with a Cell-Penetrating Peptide2007Inngår i: Mol. Ther.Artikkel i tidsskrift (Fagfellevurdert)
  • 240. Jones, Sarah
    et al.
    Holm, Tina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mäger, Imre
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Howl, John
    Characterisation of bioactive cell penetrating peptides from human Cytochrome c: protein mimicry and the development of a novel apoptogenic agent2010Inngår i: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 17, nr 7, s. 735-744Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell penetrating peptides (CPPs) with intrinsic biological activities offer a novel strategy for the modulation of intracellular events. QSAR analysis identified CPPs within human cytochrome c. Two such sequences, Cyt c77–101 and Cyt c86–101, induced tumor cell apoptosis, thus mimicking the role of Cyt c as a key regulator of programmed cell death. Quantitative analyses confirmed that Cyt c77–101 is an extremely efficient CPP. Thus, Cyt c77–101 was selected for modification to incorporate target-specific peptidyl motifs. Chimeric N-terminal extension with a target mimetic of FG nucleoporins significantly enhanced the apoptogenic potency of Cyt c77–101 to a concentration readily achievable in vivo. Moreover, this construct, Nup153-Cyt c, facilitates the dramatic redistribution of nuclear pore complex proteins and thus propounds the nuclear pore complex as a novel target for the therapeutic induction of apoptosis.

  • 241. Jones, Sarah
    et al.
    Lukanowska, Monika
    Suhorutsenko, Julia
    Oxenham, Senga
    Barratt, Christopher
    Publicover, Steven
    Copolovici, Dana Maria
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Howl, John
    Intracellular translocation and differential accumulation of cell-penetrating peptides in bovine spermatozoa: evaluation of efficient delivery vectors that do not compromise human sperm motility2013Inngår i: Human Reproduction, ISSN 0268-1161, E-ISSN 1460-2350, Vol. 28, nr 7, s. 1874-1889Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Do cell penetrating peptides (CPPs) translocate into spermatozoa and, if so, could they be utilized to deliver a much larger protein cargo? Chemically diverse polycationic CPPs rapidly and efficiently translocate into spermatozoa. They exhibit differential accumulation within intracellular compartments without detrimental influences upon cellular viability or motility but they are relatively ineffective in transporting larger proteins. Endocytosis, the prevalent route of protein internalization into eukaryotic cells, is severely compromised in mature spermatozoa. Thus, the translocation of many bioactive agents into sperm is relatively inefficient. However, the delivery of bioactive moieties into mature spermatozoa could be significantly improved by the identification and utility of an efficient and inert vectorial delivery technology. CPP translocation efficacies, their subsequent differential intracellular distribution and the influence of peptides upon viability were determined in bovine spermatozoa. Temporal analyses of sperm motility in the presence of exogenously CPPs utilized normozoospermic human donor samples. CPPs were prepared by manual, automated and microwave-enhanced solid phase synthesis. Confocal fluorescence microscopy determined the intracellular distribution of rhodamine-conjugated CPPs in spermatozoa. Quantitative uptake and kinetic analyses compared the translocation efficacies of chemically diverse CPPs and conjugates of biotinylated CPPs and avidin. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) conversion assays were employed to analyse the influence of CPPs upon sperm cell viability and sperm class assays determined the impact of CPPs on motility in capacitated and non-capacitated human samples. Chemically heterogeneous CPPs readily translocated into sperm to accumulate within discrete intracellular compartments. Mitoparan (INLKKLAKL(Aib)KKIL), for example, specifically accumulated within the mitochondria located in the sperm midpiece. The unique plasma membrane composition of sperm is a critical factor that directly influences the uptake efficacy of structurally diverse CPPs. No correlations in efficacies were observed when comparing CPP uptake into sperm with either uptake into fibroblasts or direct translocation across a phosphatidylcholine membrane. These comparative investigations identified C105Y (CSIPPEVKFNKPFVYLI) as a most efficient pharmacokinetic modifier for general applications in sperm biology. Significantly, CPP uptake induced no detrimental influence upon either bovine sperm viability or the motility of human sperm. As a consequence of the lack of endocytotic machinery, the CPP-mediated delivery of much larger protein complexes into sperm is relatively inefficient when compared with the similar process in fibroblasts. It is possible that some CPPs could directly influence aspects of sperm biology and physiology that were not analysed in this study. CPP technologies have significant potential to deliver selected bioactive moieties and so could modulate the biology and physiology of human sperm biology both prior- and post-fertilization. We are pleased to acknowledge financial support from the following sources: the Wellcome Trust, TENOVUS (Scotland), University of Dundee, Medical Research Council, NHS Tayside and Scottish Enterprise and the Research Institute in Healthcare Science, University of Wolverhampton. No conflicts of interest are reported by the authors.

  • 242. Josephy, P. David
    et al.
    Pan, Dan
    Ianni, Michael D.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Functional studies of single-nucleotide polymorphic variants of human glutathione transferase T1-1 involving residues in the dimer interface2011Inngår i: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 513, nr 2, s. 87-93Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferase T1-1 catalyses detoxication and bioactivation processes in which glutathione conjugates are formed from endogenous and xenobiotic substrates, including alkylating agents and halogenated alkanes. Although the common null polymorphism of the human GSTT1 gene has been studied extensively, little is known about the consequences of GSTT1 single-nucleotide polymorphisms (SNPs). Here, we have examined the effects of two SNPs that alter amino acid residues in the dimer interface of the GST T1-1 protein and one that causes a conservative substitution in the core of the subunit. Variant proteins were expressed in an Escherichia coli strain in which the metabolism of ethylene dibromide to a glutathione conjugate leads to lacZ reversion mutations. We measured the kinetic properties of the enzymes with the characteristic substrate 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and determined the specific activities with several other substrates. Circular dichroism spectroscopy was used to measure protein thermal denaturation profiles. Variant T104P, which has been reported as inactive, showed weak but detectable activity with each substrate. Variant R76S was expressed at lower levels and showed much-reduced thermal stability. The results are interpreted in the context of the three-dimensional structure of human GST T1-1.

  • 243. Juks, Carmen
    et al.
    Lorents, Annely
    Arukuusk, Piret
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Pooga, Margus
    Cell-penetrating peptides recruit type A scavenger receptors to the plasma membrane for cellular delivery of nucleic acids2017Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 31, nr 3, s. 975-988Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Scavenger receptors (SRs) are a large family of multifunctional receptors that are involved in a range of physiologic and pathologic processes. The ability of class A scavenger receptors (SR-As) to bind anionic ligands facilitates the internalization of negatively charged cell-penetrating peptide (CPP)-nucleic acid nanocomplexes and thus makes them attractive targets for delivery of various nucleic acids. Recently, we demonstrated that SR-A3 and SR-A5 are recruited from intracellular membranes to the plasma membrane after incubation with PepFect 14-splice-switching oligonucleotide complexes. Here, we examined the mechanisms responsible for translocation of SR-As to the cell surface. We demonstrate that, in addition to nanocomplexes, some amphipathic CPPs are able to induce externalization of SR-A3 and SR-A5, and this process requires the presence of calcium ions. Furthermore, translocation of SR-A3 and SR-A5 requires activity of phosphatidylinositol-3-kinase, intact actin cytoskeleton, and the presence of serum proteins in culture medium.

  • 244. Juks, Carmen
    et al.
    Padari, Kärt
    Margus, Helerin
    Kriiska, Asko
    Etverk, Indrek
    Arukuusk, Piret
    Koppel, Kaida
    Ezzat, Kariem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Oxford, UK.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Pooga, Margus
    The role of endocytosis in the uptake and intracellular trafficking of PepFect14-nucleic acid nanocomplexes via class A scavenger receptors2015Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1848, nr 12, s. 3205-3216Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell penetrating peptides are efficient tools to deliver various bioactive cargos into cells, but their exactfunctioning mechanism is still debated. Recently, we showed that a delivery peptide PepFect14 condenses oligonudeotides (ON) into negatively charged nanocomplexes that are taken up by cells via class A scavenger receptors (SR-As). Here we unraveled the uptake mechanism and intracellular trafficking of PF14-ON nanocomplexes in HeLa cells. Macropinocytosis and caveolae-mediated endocytosis are responsible for the intracellular functionality of nucleic acids packed into nanocomplexes. However, only a negligible fraction of the complexes were trafficked to endoplasmic reticulum or Golgi apparatus the common destinations of caveolar endocytosis. Neither were the PF14-SCO nanocomplexes routed to endo-lysosomal pathway, and they stayed in vesicles with slightly acidic pH, which were not marked with LysoSensor. Naked ON, in contrary, was rapidly targeted to acidic vesicles and lysosomes. The transmission electron microscopy analysis of interactions between SR-As and PF14-ON nanocomplexes on ultrastructural level revealed that nanocomplexes localized on the plasma membrane in close proximity to SR-As and their colocalization is retained in cells, suggesting that PF14-ON complexes associate with targeted receptors.

  • 245.
    Järver, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides as delivery vectors for oligonucleotides and proteins: Studies on applications and toxicity2007Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Cell-penetrating peptides (CPPs) have for a little bit more than a decade been employed as delivery vectors for a wide range of cargoes, ranging from gold particles to entire plasmids. Although CPP are well studied and utilized in numerous publications, our knowledge about CPP mediated transport is still poor. The articles presented in this thesis all consider different aspects of CPP mediated delivery. The first two papers are evaluating and improving already known techniques. In paper I, standard polyethyleneimine (PEI) transfection is improved by conjugating the CPP TP10 to the cationic polymer. In paper II, the same CPP is employed to deliver a dsDNA decoy oligo, resulting in decreased activity of the transcription factor c-Myc. The third paper is a more general overview of the delivery efficiency of well known CPPs and how the delivered cargo influences the CPP mediated toxicity. The study shows that different CPPs are suitable for different cargos and that toxic side effects depend heavily on the cargo and coupling strategy used. In Paper IV, a novel CPP, M918, is evaluated as a delivery vector for a transposon based non-viral gene therapy system. M918 display simultaneous delivery of a plasmid carrying a selection gene and a transposase into cultured cells. This is the first study where two so vastly different molecules as a cationic protein and an anionic plasmid, are simultaneously transported into cells by a peptide vector. The method might be a first step towards a safe peptide based non-viral gene therapy platform. Taken together, the results presented in this thesis might help to improve already existing techniques, increase our understandings about CPP mediated delivery and, at the same time, develop new CPP based delivery systems.

  • 246.
    Järver, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Fernaeus, Sandra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Tjörnhammar, Marie-Louise
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Co-transduction of Sleeping Beauty Transposase and Donor Plasmid via a Cell-penetrating Peptide: A simple one-step method2008Inngår i: International journal of peptide research and therapeutics, ISSN 1573-3904, Vol. 14, nr 1, s. 58-63Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Transposable elements have emerged as a promising candidate for human non-viral gene-therapy. The Tc1/mariner transposon Sleeping Beauty is to date one of the most efficient transposons in mammals. Sleeping Beauty transposase has so far mostly been delivered to cells via a DNA source. This might cause spontaneous integration of the transposase gene and cause fatal damage to the affected cell. Hence, it would be advantageous to employ a non-genetic source for the transposase. We here show that a novel Cell-penetrating peptide, M918, has the ability to facilitate cellular delivery of both the transposase Sleeping Beauty as a protein and a transposon donor-plasmid carrying an antibiotic resistance gene in vitro. The technique is a simple and straightforward one-step method that might render a safe and efficient delivery platform for Sleeping Beauty mediated gene therapy.

  • 247.
    Järver, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, K.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Applications of cell-penetrating peptides in regulation of gene expression2007Inngår i: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 35, nr Pt 4, s. 770-774Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    CPPs (cell-penetrating peptides) can be defined as short peptides that are able to efficiently penetrate cellular lipid bilayers. Because of this remarkable feature, they are excellent candidates regarding alterations in gene expression. CPPs have been utilized in in vivo and in vitro experiments as delivery vectors for different bioactive cargoes. This review focuses on the experiments performed in recent years where CPPs have been used as vectors for multiple effectors of gene expression such as oligonucleotides for antisense, siRNA (small interfering RNA) and decoy dsDNA (double-stranded DNA) applications, and as transfection agents for plasmid delivery.

  • 248. Järver, Peter
    et al.
    Mäger, Imre
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia .
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    In vivo biodistribution and efficacy of peptide mediated delivery2010Inngår i: TIPS - Trends in Pharmacological Sciences, ISSN 0165-6147, E-ISSN 1873-3735, Vol. 31, nr 11, s. 528-535Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    To transverse the plasma membrane and gain access to the cellular interior is one of the major obstacles for many novel pharmaceutical molecules. Since the late 1990 s, cell-penetrating peptides (CPPs) have been utilized as transport vectors for a broad spectrum of ‘biological cargoes’, ranging from inert gold particles to multifaceted macromolecules such as proteins and plasmids. Numerous studies have shown that CPPs are efficient carriers for bioactive cargoes in vitro. However, even though CPPs are versatile transport vectors, this does not guarantee they can be developed into useful pharmaceutical molecules. Nevertheless, recent progress in the field has shown CPPs to be effective for in vivo delivery with retained biological activity of a wide variety of bioactive cargoes into virtually any mammalian tissue. This review will focus on recent developments and applications for CPP delivery and distribution in vivo.

  • 249.
    Järver, Peter
    et al.
    Medical Research Council , Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom.
    Zaghloul, Eman M
    Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Huddinge, Sweden.
    Arzumanov, Andrey A
    Medical Research Council, Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom.
    Saleh, Amer F
    Medical Research Council, Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom.
    McClorey, Graham
    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
    Hammond, Suzan M
    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
    Hällbrink, Mattias
    Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Huddinge, Sweden.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Smith, C I Edvard
    Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Huddinge, Sweden.
    Wood, Matthew J A
    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
    Gait, Michael J
    Medical Research Council, Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom.
    El Andaloussi, Samir
    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
    Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.2015Inngår i: Nucleic Acid Therapeutics, ISSN 2159-3337, E-ISSN 2159-3345, Vol. 25, nr 2, s. 65-77Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.

  • 250. Kappe, Camilla
    et al.
    Tracy, Linda M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Patrone, Cesare
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sjöholm, Åke
    GLP 1 secretion by microglial cells and decreased cns expression in obesity2012Inngår i: Journal of Neuroinflammation, ISSN 1742-2094, E-ISSN 1742-2094, Vol. 9, s. 276-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Type 2 diabetes (T2D) is a strong risk factor for developing neurodegenerative pathologies. T2D patients have a deficiency in the intestinal incretin hormone GLP-1, which has been shown to exert neuroprotective and anti-inflammatory properties in the brain. Methods: Here we investigate potential sources of GLP-1 in the CNS and the effect of diabetic conditions on the proglucagon mRNA expression in the CNS. The obese mouse model ob/ob, characterized by its high levels of free fatty acids, and the microglia cell line BV-2 were used as models. mRNA expression and protein secretion were analyzed by qPCR, immunofluorescence and ELISA. Results: We show evidence for microglia as a central source of GLP-1 secretion. Furthermore, we observed that expression and secretion are stimulated by cAMP and dependent on microglial activation state. We also show that insulin-resistant conditions reduce the central mRNA expression of proglucagon. Conclusion: The findings that microglial mRNA expression of proglucagon and GLP-1 protein expression are affected by high levels of free fatty acids and that both mRNA expression levels of proglucagon and secretion levels of GLP-1 are affected by inflammatory stimuli could be of pathogenic importance for the premature neurodegeneration and cognitive decline commonly seen in T2D patients, and they may also be harnessed to advantage in therapeutic efforts to prevent or treat such disorders.

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