Change search
Refine search result
12 1 - 50 of 99
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1. Ali, Raja Hashim
    et al.
    Muhammad, Sayyed Auwn
    Khan, Mehmood Alam
    Arvestad, Lars
    Stockholm University, Faculty of Science, Numerical Analysis and Computer Science (NADA). Stockholm University, Science for Life Laboratory (SciLifeLab). Swedish e-Science Research Center, Sweden .
    Quantitative synteny scoring improves homology inference and partitioning of gene families2013In: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 14, no Suppl,15, p. S12-Article in journal (Refereed)
    Abstract [en]

    Background

    Clustering sequences into families has long been an important step in characterization of genes and proteins. There are many algorithms developed for this purpose, most of which are based on either direct similarity between gene pairs or some sort of network structure, where weights on edges of constructed graphs are based on similarity. However, conserved synteny is an important signal that can help distinguish homology and it has not been utilized to its fullest potential.

    Results

    Here, we present GenFamClust, a pipeline that combines the network properties of sequence similarity and synteny to assess homology relationship and merge known homologs into groups of gene families. GenFamClust identifies homologs in a more informed and accurate manner as compared to similarity based approaches. We tested our method against the Neighborhood Correlation method on two diverse datasets consisting of fully sequenced genomes of eukaryotes and synthetic data.

    Conclusions

    The results obtained from both datasets confirm that synteny helps determine homology and GenFamClust improves on Neighborhood Correlation method. The accuracy as well as the definition of synteny scores is the most valuable contribution of GenFamClust.

  • 2.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The Mitochondrial Peptidasome, PreP, relation to Alzheimer Disease2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Amyloid-β (Aβ) is the toxic peptide implicated in the pathogenesis of Alzheimer Disease (AD). Accumulation of Aβ has been shown in brain mitochondria from AD patients and AD mice models. The occurrence of Aβ in the mitochondrial matrix leads to free radical generation and apoptosis in neurons.

    In our studies, Aβ was found in brain mitochondria of living patients with plaque pathology. Extracellular Aβ was taken up by neuroblastoma cells and was found in the mitochondria. Moreover, we showed that Aβ40 and Aβ42 are transported into mitochondria via the Translocase of the Outer Membrane, the TOM machinery.

    We have identified the mitochondrial Aβ-degrading protease hPreP, in human brain mitochondrial matrix. PreP is a metalloprotease, originally identified as presequence protease, but was also shown to degrade other unstructured peptides including Aβ. Immunoinactivation of PreP in human brain mitochondria revealed PreP to be the protease responsible for Aβ degradation in mitochondria.

    Also, we have investigated if genetic variation in the gene encoding hPreP is associated with AD by genotyping 19 single nucleotide polymorphisms (SNPs) in the Swedish population. The study did not show a genetic association between any of the genotyped SNPs and the risk to AD. However, the biochemical analysis of four SNPs selected on the basis of their location within a structural homology model of hPreP revealed a decreased activity compared to wildtype.

    Interestingly, the activity of PreP in human brain mitochondrial matrix in AD individuals was significantly lower compared to non-dement aged-matched controls. These findings were also confirmed in brain mitochondrial matrix of AD mouse models. These results suggest that a decreased PreP activity may contribute to Aβ aggregation and accumulation inside mitochondria leading to neuronal death in AD. In summary, our findings show that the degradation of Aβ by hPreP may be of importance in the pathology of AD.

  • 3.
    Baars, Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Protein targeting, translocation and insertion in Escherichia coli: Proteomic analysis of substrate-pathway relationships2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Approximately 10% of the open reading frames in the genome of the Gram-negative bacterium E. coli encodes secretory proteins, and 20% encodes integral inner membrane proteins (IMPs). These proteins are sorted to their correct cellular compartments (the periplasm and the outer and inner membranes) by specialized targeting and translocation/insertion systems. So far, a very limited set of model proteins have been used to study proteins sorting requirements in E. coli. The main objective of all the papers presented in this thesis was to determine the targeting and translocation/insertion requirements of more E. coli proteins. In papers I and II, this was done using focused approaches. Selected model proteins (lipoproteins and putative outer membrane proteins) were expressed from plasmids and their targeting and translocation were analysed in vitro by crosslinking experiments and/or in vivo by pulse-chase analysis in different E. coli mutant strains. In papers III a comparative sub-proteome analysis was carried out to define the role of the cytoplasmic chaperone SecB in protein targeting. In paper IV, a similar approach was used to study how protein translocation and insertion is affected upon depletion of the essential Sec-translocon component SecE. The ‘global’ approach used in paper III and IV allowed us to study protein targeting and translocation/insertion requirements on a proteome level. This led to the identification of several novel SecB substrates and a large number of potential Sec-translocon independent IMPs.

  • 4.
    Bajinskis, Ainars
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Natarajan, Adayapalam T.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Harms-Ringdahl, Mats
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    DNA double strand breaks induced by the indirect effect of radiation are more efficiently repaired by non-homologous end joining compared to homologous recombination repair2013In: Mutation research. Genetic toxicology and environmental mutagenesis, ISSN 1383-5718, E-ISSN 1879-3592, Vol. 756, no 1-2, p. 21-29Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to investigate the relative involvement of three major DNA repair pathways, i.e., non-homologous end joining (NHEJ), homologous recombination (HRR) and base excision (BER) in repair of DNA lesions of different complexity induced by low- or high-LET radiation with emphasis on the contribution of the indirect effect of radiation for these radiation qualities. A panel of DNA repair-deficient CHO cell lines was irradiated by Cs-137 gamma-rays or radon progeny alpha-particles. Irradiation was also performed in the presence of 2 M DMSO to reduce the indirect effect of radiation and the complexity of the DNA damage formed. Clonogenic survival and micronucleus assays were used to estimate efficiencies of the different repair pathways for DNA damages produced by direct and indirect effects. Removal of the indirect effect of low-LET radiation by DMSO increased clonogenic survival and decreased MN formation for all cell lines investigated. A direct contribution of the indirect effect of radiation to DNA base damage was suggested by the significant protection by DMSO seen for the BER deficient cell line. Lesions formed by the indirect effect are more readily repaired by the NHEJ pathway than by HRR after irradiation with gamma-rays or alpha-particles as evaluated by cell survival and the yields of MN. The results obtained with BER- and NHEJ-deficient cells suggest that the indirect effect of radiation contributes significantly to the formation of repair substrates for these pathways.

  • 5.
    Bakali, Amin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Herman, Maria Dolores
    Johnson, Kenneth A.
    Kelly, Amélie A.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hallberg, B. M.
    Nordlund, Pär
    Crystal structure of YegS, a homologue to the mammalian diacylglycerol kinases, reveals a novel regulatory metal binding site2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 27, p. 19644-19652Article in journal (Refereed)
    Abstract [en]

    The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.

  • 6.
    Baumgarten, Thomas
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schlegel, Susan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wagner, Samuel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Löw, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, Jonas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bonde, Ida
    Herrgård, Markus J.
    Heipieper, Hermann J.
    Nørholm, Morten H. H.
    Slotboom, Dirk Jan
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 45089Article in journal (Refereed)
    Abstract [en]

    Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production.

  • 7.
    Berglund, Anna-Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dual Targeting of Proteins to Mitochondria and Chloroplasts2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The vast majority of mitochondrial and chloroplastic proteins are nuclear encoded, synthesized in the cytosol and imported into the respective organelle using an N-terminal extension, the targeting peptide (TP). After import into the organelle, the TP is cleaved off and degraded by the Presequence protease (PreP). The import process is thought to be highly specific, however there is a group of proteins that are localised to both mitochondria and chloroplasts, using an ambiguous, dual targeting peptide (dTP). The aim of this thesis was to investigate targeting properties of dTPs. Analysis of the amino acid content of all currently known dually targeted proteins revealed that the dTPs are enriched in hydroxylated, hydrophobic and positively charged residues, lacking acidic residues, whereas the content of serine, arginine and proline is intermediary in comparison to the mitochondrial and chloroplastic TPs. dTPs do not form amphiphilic a-helices, characteristic of the mitochondrial TPs, but the helical structure can be induced in membrane mimetic environment, as revealed by spectroscopic studies of a dTP of an aminoacyl- tRNA-synthetase (aaRS). In vitro and in vivo import experiments of fusion constructs containing N-terminal truncations of seven aaRS-dTPs coupled to green fluorescent protein (GFP) demonstrated different organisation of targeting determinants showing that the N-terminal portion of dTPs was crucial for import into both organelles or at least one organelle for different constructs. In addition, studies of targeting capacity of the TPs of PreP homologues from plant, mammal and yeast (AtPreP, hPreP and Mop112) showed species dependent intra-mitochondrial localisation of the coupled GFP and demonstrated functional complementation of an intermembrane space located Mop112 with a matrix located AtPreP. The studies presented here contribute to understanding of the intracellular and intra-mitochondrial sorting process of proteins in the eukaryotic cell.

  • 8.
    Bhushan, Shashi
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kuhn, Claus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berglund, Anna-Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Roth, Christian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting2006In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 16, p. 3966-3972Article in journal (Refereed)
    Abstract [en]

    We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of Arabidopsis thaliana. Despite overall similarities, the first 16 residues of transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria in vitro and in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting.

  • 9.
    Bhushan, Shashi
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pavlov, Pavel F
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rudhe, Charlotta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    In vitro and in vivo methods to study protein import into plant mitochondria.2007In: Methods Mol Biol, ISSN 1064-3745, Vol. 390, p. 131-50Article in journal (Refereed)
    Abstract [en]

    Plant mitochondria contain about 1000 proteins, 90-99% of which in different plant species are nuclear encoded, synthesized on cytosolic polyribosomes, and imported into the organelle. Most of the nuclear-encoded proteins are synthesized as precursors containing an N-terminal extension called a presequence or targeting peptide that directs the protein to the mitochondria. Here we describe in vitro and in vivo methods to study mitochondrial protein import in plants. In vitro synthesized precursor proteins can be imported in vitro into isolated mitochondria (single organelle import). However, missorting of chloroplast precursors in vitro into isolated mitochondria has been observed. A novel dual import system for simultaneous import of proteins into isolated mitochondria and chloroplasts followed by reisolation of the organelles is superior over the single import system as it abolishes the mistargeting. Precursor proteins can also be imported into the mitochondria in vivo using an intact cellular system. In vivo approaches include import of transiently expressed fusion constructs containing a presequence or a full-length precursor protein fused to a reporter gene, most commonly the green fluorescence protein (GFP) in protoplasts or in an Agrobacterium-mediated system in intact tobacco leaves.

  • 10. Bjorklund, Geir
    et al.
    Stejskal, Vera
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Urbina, Mauricio A.
    Dadar, Maryam
    Chirumbolo, Salvatore
    Mutter, Joachim
    Metals and Parkinson's Disease: Mechanisms and Biochemical Processes2018In: Current Medicinal Chemistry, ISSN 0929-8673, E-ISSN 1875-533X, Vol. 25, no 19, p. 2198-2214Article, review/survey (Refereed)
    Abstract [en]

    Genetic background accounts for only 5 to 10% of the reported cases of Parkinson's disease (PD), while the remaining cases are of unknown etiology. It is believed that environmental factors may be involved in the causality of a large proportion of PD cases. Several PD genes are activated by xenobiotic exposure, and a link between pesticide exposure and PD has been demonstrated. Many epidemiological studies have shown an association between PD and exposure to metals such as mercury, lead, manganese, copper, iron, aluminum, bismuth, thallium, and zinc. This review explores the biological effects, the pathogenetic processes, genetic susceptibilities to metals as well as examining future strategies for PD treatment, such as chelation therapy.

  • 11.
    Björkander, Sophia
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hell, Lena
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Johansson, Maria A.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mata Forsberg, Manuel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lasaviciute, Gintare
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Roos, Stefan
    Holmlund, Ulrika
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sverremark-Ekström, Eva
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Staphylococcus aureus-derived factors induce IL-10, IFN-gamma and IL-17A-expressing FOXP3(+)CD161(+) T-helper cells in a partly monocyte-dependent manner2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 22083Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus (S. aureus) is a human pathogen as well as a frequent colonizer of skin and mucosa. This bacterium potently activates conventional T-cells through superantigens and it is suggested to induce T-cell cytokine-production as well as to promote a regulatory phenotype in T-cells in order to avoid clearance. This study aimed to investigate how S. aureus impacts the production of regulatory and pro-inflammatory cytokines and the expression of CD161 and HELIOS by peripheral CD4(+)FOXP3(+) T-cells. Stimulation of PBMC with S. aureus 161:2-cell free supernatant (CFS) induced expression of IL-10, IFN-gamma and IL-17A in FOXP3(+) cells. Further, CD161 and HELIOS separated the FOXP3(+) cells into four distinct populations regarding cytokine-expression. Monocyte-depletion decreased S. aureus 161:2-induced activation of FOXP3(+) cells while pre-stimulation of purified monocytes with S. aureus 161:2-CFS and subsequent co-culture with autologous monocyte-depleted PBMC was sufficient to mediate activation of FOXP3(+) cells. Together, these data show that S. aureus potently induces FOXP3(+) cells and promotes a diverse phenotype with expression of regulatory and pro-inflammatory cytokines connected to increased CD161-expression. This could indicate potent regulation or a contribution of FOXP3(+) cells to inflammation and repression of immune-suppression upon encounter with S. aureus.

  • 12.
    Björklund, Asa K
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Light, Sara
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hedin, Linnea
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Quantitative assessment of the structural bias in protein-protein interaction assays.2008In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 8, no 22, p. 4657-46667Article in journal (Refereed)
    Abstract [en]

    With recent publications of several large-scale protein-protein interaction (PPI) studies, the realization of the full yeast interaction network is getting closer. Here, we have analysed several yeast protein interaction datasets to understand their strengths and weaknesses. In particular, we investigate the effect of experimental biases on some of the protein properties suggested to be enriched in highly connected proteins. Finally, we use support vector machines (SVM) to assess the contribution of these properties to protein interactivity. We find that protein abundance is the most important factor for detecting interactions in tandem affinity purifications (TAP), while it is of less importance for Yeast Two Hybrid (Y2H) screens. Consequently, sequence conservation and/or essentiality of hubs may be related to their high abundance. Further, proteins with disordered structure are over-represented in Y2H screens and in one, but not the other, large-scale TAP assay. Hence, disordered regions may be important both in transient interactions and interactions in complexes. Finally, a few domain families seem to be responsible for a large part of all interactions. Most importantly, we show that there are method-specific biases in PPI experiments. Thus, care should be taken before drawing strong conclusions based on a single dataset.

  • 13.
    Björklund, Åsa K.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ekman, Diana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Expansion of Protein Domain Repeats2006In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 2, no 8, p. 959-970Article in journal (Refereed)
    Abstract [en]

    Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein-protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e. g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.

  • 14. Brechmann, Nils A.
    et al.
    Eriksson, Per-Olov
    Eriksson, Kristofer
    Oscarsson, Sven
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Buijs, Jos
    Shokri, Atefeh
    Hjälm, Göran
    Chotteau, Véronique
    Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture2019In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 35, no 3, article id e2775Article in journal (Refereed)
    Abstract [en]

    High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25-42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 x 10(6) cells/mL as in clarified supernatant. Two pilot-scale purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L, where a rapid mAb adsorption >= 96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient single step, which directly connected the culture to the downstream process without cell clarification. Purification of mAb directly from non-clarified cell broth without cell separation can provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.

  • 15.
    Brändén, Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Electron and proton transfer in cytochrome c oxidase2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This doctoral thesis describes results from studies on the mechanisms by which the enzyme cytochrome c oxidase catalyses the reduction of oxygen to water. Cytochrome c oxidase is the last integral enzyme complex of the socalled respiratory chain in the mitochondrial inner membrane (plasma membrane of bacteria). The reaction catalysed by cytochrome c oxidase produces a charge separation across the membrane by two separate mechanisms. The protons and the electrons needed to reduce oxygen to water are supplied from different sides of the membrane, and the energy released in this reaction is used by cytochrome c oxidase to pump protons against the electro-chemical gradient across the membrane. Another integral enzyme, ATP-synthase, utilises this gradient for synthesis of ATP, which in turn is used in many of the energyrequiring processes of a cell. Cytochrome c oxidase holds four redox-active metal centres that mediate electron transfer to, and reduction of the O2-molecule. One part of the thesis concerns the rates of electron transfer as well as the redox equilibrium between the metal sites (papers II and IV).

    Since the oxygen chemistry takes place in the membrane-spanning part of the enzyme it is equipped with two proton-transfer pathways that lead from the bulk solution to the active site. A second part of the thesis concerns the location of the entry-point of one of these pathways as well as the role of this pathway during the catalytic cycle (papers I and III).

    The third part of the thesis concerns the mechanism by which the enzymecouples the O2-chemistry to proton pumping (paper V).

  • 16.
    Bäckman, Hans G
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pessoa, João
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eneqvist, Therese
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Binding of divalent cations is essential for the activity of the organellar peptidasome in Arabidopsis thaliana, AtPreP.2009In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 583, no 17, p. 2727-33Article in journal (Refereed)
    Abstract [en]

    The dual-targeted mitochondrial and chloroplastic zinc metallooligopeptidase from Arabidopsis, AtPreP, functions as a peptidasome that degrades targeting peptides and other small unstructured peptides. In addition to Zn located in the catalytic site, AtPreP also contains two Mg-binding sites. We have investigated the role of Mg-binding using AtPreP variants, in which one or both sites were rendered unable to bind Mg(2+). Our results show that metal binding besides that of the active site is crucial for AtPreP proteolysis, particularly the inner site appears essential for normal proteolytic function. This is also supported by its evolutionary conservation among all plant species of PreP.

  • 17. Carter, Victoria
    et al.
    Underhill, Ann
    Baber, Ibrahima
    Sylla, Lakamy
    Baby, Mounirou
    Larget-Thiery, Isabelle
    Zettor, Agnès
    Bourgouin, Catherine
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Otvos, Laszlo
    Wade, John D.
    Coulibaly, Mamadou B.
    Traore, Sekou F.
    Tripet, Frederic
    Eggleston, Paul
    Hurd, Hilary
    Killer bee molecules: antimicrobial peptides as effector molecules to target sporogonic stages of Plasmodium2013In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 9, no 11, article id e1003790Article in journal (Refereed)
    Abstract [en]

    A new generation of strategies is evolving that aim to block malaria transmission by employing genetically modified vectors or mosquito pathogens or symbionts that express anti-parasite molecules. Whilst transgenic technologies have advanced rapidly, there is still a paucity of effector molecules with potent anti-malaria activity whose expression does not cause detrimental effects on mosquito fitness. Our objective was to examine a wide range of antimicrobial peptides (AMPs) for their toxic effects on Plasmodium and anopheline mosquitoes. Specifically targeting early sporogonic stages, we initially screened AMPs for toxicity against a mosquito cell line and P. berghei ookinetes. Promising candidate AMPs were fed to mosquitoes to monitor adverse fitness effects, and their efficacy in blocking rodent malaria infection in Anopheles stephensi was assessed. This was followed by tests to determine their activity against P. falciparum in An. gambiae, initially using laboratory cultures to infect mosquitoes, then culminating in preliminary assays in the field using gametocytes and mosquitoes collected from the same area in Mali, West Africa. From a range of 33 molecules, six AMPs able to block Plasmodium development were identified: Anoplin, Duramycin, Mastoparan X, Melittin, TP10 and Vida3. With the exception of Anoplin and Mastoparan X, these AMPs were also toxic to an An. gambiae cell line at a concentration of 25 µM. However, when tested in mosquito blood feeds, they did not reduce mosquito longevity or egg production at concentrations of 50 µM. Peptides effective against cultured ookinetes were less effective when tested in vivo and differences in efficacy against P. berghei and P. falciparum were seen. From the range of molecules tested, the majority of effective AMPs were derived from bee/wasp venoms.

  • 18.
    Chiruvella, Kishore K.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Rajaei, Naghmeh
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Jonna, Venkateswara Rao
    Hofer, Anders
    Åström, Stefan U.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 21671Article in journal (Refereed)
    Abstract [en]

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MAT alpha). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.

  • 19.
    Dian, Cyril
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Adaptive Responses by Transcriptional Regulators to small molecules in Prokaryotes: Structural studies of two bacterial one-component signal transduction systems DntR and HpNikR2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Prokaryotes are continually exposed to variations in their environment. Survival in unstable milieu requires a wide range of transcriptional regulators (TRs) that respond to specific environmental and cellular signals by modulating gene expression and provide an appropriate physiological response to external stimuli. These adaptive responses to environmental signals are mostly mediated by TRs from one of two families: the single or the two component signal transduction systems (1CSTS; 2CSTS). In this thesis the structural analysis of two 1CSTS – DntR and NikR − are presented. One study was carried out to try to develop a bacterial biosensor for synthetic dinitrotulenes compounds, the other to characterise the Ni-sensing mechanism that contributes to the acid adaptation of the human pathogen Helicobacter pylori. DntR belongs to the LysR family and the crystal structures obtained have allowed the proposal a model of the interaction of DntR with salicylate inducer as well as giving insights into the signal propagation mechanism in LysR-type transcription factors (paper I). DntR mutant crystal structures combined with the modelling of DntR-2,4-dnt interactions led to the design of a DntR mutant that has a limited response to 2,4-dnt in a whole cell biosensor system (paper 2). Crystal structures of apo-NikR from H. pylori (HpNikR) and of Ni-bound intermediary states of the protein were obtained. The latter have helped in unravelling the Ni incorporation and selectivity mechanisms of NikRs and have shown a strong cooperativity between conformational changes in the Ni binding domain with movements of the DNA binding domain (paper 3). Biochemical studies and comparisons of the HpNikR crystal structures with those of NikR homologues strongly suggest that HpNikR has evolved different surface properties (paper 4) and a new mode of DNA binding.

  • 20.
    Eriksson, Olaspers Sara
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Geörg, Miriam
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sjölinder, Hong
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sillard, Rannar
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jonsson, Ann-Beth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Identification of Cell-Penetrating Peptides That Are Bactericidal to Neisseria meningitidis and Prevent Inflammatory Responses upon Infection2013In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 57, no 8, p. 3704-3712Article in journal (Refereed)
    Abstract [en]

    Meningococcal disease is characterized by a fast progression and a high mortality rate. Cell-penetrating peptides (CPPs), developed as vectors for cargo delivery into eukaryotic cells, share structural features with antimicrobial peptides. A screen identified two CPPs, transportan-10 (TP10) and model amphipathic peptide (MAP), with bactericidal action against Neisseria meningitidis. Both peptides were active in human whole blood at micromolar concentrations, while hemolysis remained negligible. Additionally, TP10 exhibited significant antibacterial activity in vivo. Uptake of SYTOX green into live meningococci was observed within minutes after TP10 treatment, suggesting that TP10 may act by membrane permeabilization. Apart from its bactericidal activity, TP10 suppressed inflammatory cytokine release from macrophages infected with N. meningitidis as well as from macrophages stimulated with enterobacterial and meningococcal lipopolysaccharide (LPS). Finally, incubation with TP10 reduced the binding of LPS to macrophages. This novel endotoxin-inhibiting property of TP10, together with its antimicrobial activity in vivo, indicates the possibility to design peptide-based therapies for infectious diseases.

  • 21.
    Eriksson, Olivia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brinne, Björn
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zhou, Yishao
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Björkegren, Johan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tegnér, Jesper
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Deconstructing the core dynamics from a complex time-lagged regulatory biological circuit2009In: IET systems biology, ISSN 1751-8849, Vol. 3, no 2, p. 113-129Article in journal (Refereed)
    Abstract [en]

    Complex regulatory dynamics is ubiquitous in molecular networks composed of genes and proteins. Recent progress in computational biology and its application to molecular data generate a growing number of complex networks. Yet, it has been difficult to understand the governing principles of these networks beyond graphical analysis or extensive numerical simulations. Here the authors exploit several simplifying biological circumstances which thereby enable to directly detect the underlying dynamical regularities driving periodic oscillations in a dynamical nonlinear computational model of a protein-protein network. System analysis is performed using the cell cycle, a mathematically well-described complex regulatory circuit driven by external signals. By introducing an explicit time delay and using a -tearing-and-zooming- approach the authors reduce the system to a piecewise linear system with two variables that capture the dynamics of this complex network. A key step in the analysis is the identification of functional subsystems by identifying the relations between state-variables within the model. These functional subsystems are referred to as dynamical modules operating as sensitive switches in the original complex model. By using reduced mathematical representations of the subsystems the authors derive explicit conditions on how the cell cycle dynamics depends on system parameters, and can, for the first time, analyse and prove global conditions for system stability. The approach which includes utilising biological simplifying conditions, identification of dynamical modules and mathematical reduction of the model complexity may be applicable to other well-characterised biological regulatory circuits.

  • 22.
    Esbjörner, Elin K.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oglęcka, Kamila
    Lincoln, Per
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordén, Bengt
    Membrane binding of pH-sensitive Influenza fusion peptides. Positioning, configuration and induced leakage in lipid vesicles models2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 47, p. 13490-13504Article in journal (Refereed)
    Abstract [en]

    pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA 1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion (∼60-65° relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue

  • 23.
    Ezzat, Kariem
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cell-penetrating peptides; chemical modification, mechanism of uptake and formulation development2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides (CPPs) that have superior delivery properties for several nucleic acid-based therapeutics are developed. These CPPs can spontaneously form nanoparticles upon non-covalent complexation with the nucleic acid cargo, and the formed nanoparticles mediate efficient cellular transfection. In paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (PF3) can efficiently transfect different cell types with plasmid DNA and mediates efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, a new peptide with ornithine modification, PF14, is shown to efficiently deliver splice-switching oligonucleotides (SSOs) in different cell models including mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Additionally, we describe a method for incorporating the PF14-SSO nanoparticles into a solid formulation that is active and stable even when stored at elevated temperatures for several weeks. In paper III, we demonstrate the involvement of class-A scavenger receptor subtypes (SCARA3 & SCARA5) in the uptake of PF14-SSO nanoparticles, which possess negative surface charge, and suggest for the first time that some CPP-based systems function through scavenger receptors. In paper IV, the ability of PF14 to deliver siRNA to different cell lines is shown and their stability in simulated gastric acidic conditions is highlighted.

    Taken together, these results demonstrate that certain chemical modifications can drastically enhance the activity and stability of CPPs for delivering nucleic acids after spontaneous nanoparticle formation upon non-covalent complexation. Moreover, we show that CPP-based nanoparticles can be formulated into convenient and stable solid formulations that can be suitable for several therapeutic applications. Importantly, the involvement of scavenger receptors in the uptake of such nanoparticles is presented, which could yield novel possibilities to understand and improve the transfection by CPPs and other gene therapy nanoparticles.

  • 24.
    Ezzat, Kariem
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Pernemalm, Maria
    Pålsson, Sandra
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Roberts, Thomas C.
    Järver, Peter
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Dondalska, Aleksandra
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Bestas, Burcu
    Sobkowiak, Michal J.
    Levanen, Bettina
    Skold, Magnus
    Thompson, Elizabeth A.
    Saher, Osama
    Kari, Otto K.
    Lajunen, Tatu
    Ekström, Eva Sverremark
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nilsson, Caroline
    Ishchenko, Yevheniia
    Malm, Tarja
    Wood, Matthew J. A.
    Power, Ultan F.
    Masich, Sergej
    Linden, Anders
    Sandberg, Johan K.
    Lehtio, Janne
    Spetz, Anna-Lena
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    EL Andaloussi, Samir
    The viral protein corona directs viral pathogenesis and amyloid aggregation2019In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 2331Article in journal (Refereed)
    Abstract [en]

    Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid beta-peptide (A beta(42)), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.

  • 25.
    Ezzat, Kariem
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Zaghloul, Eman M.
    EL Andaloussi, Samir
    Lehto, Taavi
    Hilal, Ramy
    Magdy, Tarek
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Smith, Edvard C. I.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Solid formulation of cell-penetrating peptide nanoparticles with siRNA and their stability in simulated gastric conditions2012In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 162, no 1, p. 1-8Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are short cationic peptides that have been extensively studied as drug delivery vehicles for proteins, nucleic acids and nanoparticles. However, the formulation of CPP-based therapeutics into different pharmaceutical formulations and their stability in relevant biological environments have not been given the same attention. Here, we show that a newly developed CPP, PepFect 14 (PF14), forms non-covalent nanocomplexes with short interfering RNA (siRNA), which are able to elicit efficient RNA-interference (RNAi) response in different cell-lines. RNAi effect was obtained at low siRNA doses with a unique kinetic profile. Furthermore, we utilized the solid dispersion technique to formulate PF14/siRNA nanocomplexes into solid formulations that were as active as the freshly prepared nanocomplexes in solution. Importantly, the freshly prepared nanocomplexes and solid formulations were stable after incubation with simulated gastric fluid having a pH of 1.2 and containing proteolytic enzymes. These results demonstrate the activity of PF14 in delivering and protecting siRNA in different pharmaceutical forms and biological environments.

  • 26. Faxén, Kristina
    et al.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The inside pH determines rates of electron and proton transfer in vesicle-reconstituted cytochrome c oxidase.2007In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1767, no 5, p. 381-386Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6–9.5).

  • 27. Freimann, Krista
    et al.
    Arukuusk, Piret
    Kurrikoff, Kaido
    Pärnaste, Ly
    Raid, Raivo
    Piirsoo, Andres
    Pooga, Margus
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Formulation of Stable and Homogeneous Cell-Penetrating Peptide NF55 Nanoparticles for Efficient Gene Delivery In Vivo2018In: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, E-ISSN 2162-2531, Vol. 10, p. 28-35Article in journal (Refereed)
    Abstract [en]

    Although advances in genomics and experimental gene therapy have opened new possibilities for treating otherwise incurable diseases, the transduction of nucleic acids into the cells and delivery in vivo remain challenging. The high molecular weight and anionic nature of nucleic acids require their packing into nanoparticles for the delivery. The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but their distribution in organisms is mostly governed by the physical properties of nanoparticles, and therefore, generation of stable particles with strictly defined characteristics is highly essential. Using previously designed efficient cell-penetrating peptide NF55, we searched for strategies enabling control over the nanoparticle formation and properties to further improve transfection efficacy. The size of the NF55/pDNA nanoparticles correlates with the concentration of its constituents at the beginning of assembly, but characteristics of nanoparticles measured by DLS do not reliably predict the applicability of particles in in vivo studies. We introduce a new formulation approach called cryo-concentration, where we acquired stable and homogeneous nanoparticles for administration in vivo. The cryo-concentrated NF55/pDNA nanoparticles exhibit several advantages over standard formulation: They have long shelf-life and do not aggregate after reconstitution, have excellent stability against enzymatic degradation, and show significantly higher bioactivity in vivo.

  • 28.
    Garcia Lobato Tavares, Raquel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Host cell responses to Helicobacter pylori secreted factors2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The infection of the human gastric mucosa by the bacterium Helicobacter pylori can lead to the development of gastritis, gastroduodenal ulcers, and cancer. The factors that determine disease development in a small percentage of infected individuals are still not fully understood.

    In this thesis, we aimed to identify and functionally characterize novel virulence factors of H. pylori and to understand their effect on host cell responses.

    In Paper I, we found that JHP0290, an uncharacterized secreted protein of H. pylori, induced macrophage apoptosis concomitant to the release of pro-inflammatory cytokine TNF via the regulation of the Src family of kinases and ERK MAPK pathways. In paper II, we demonstrated that JHP0290 exhibits both proliferative and anti-apoptotic activity, together with a faster progression of the cell cycle in gastric epithelial cells. During these responses, ERK MAPK and NF-κB pathways were activated. Paper III revealed a pro-apoptotic effect of another H. pylori-secreted protein HP1286 in macrophages via the TNF-independent and ERK-dependent pathways. No apoptosis was observed in HP1286-treated T cells or HL60 neutrophil-like cells, suggesting cell-type specific effect of HP1286. In Paper IV, we observed the pro-inflammatory activity of H. pylori secreted protein HP1173 in macrophages. The protein was found to induce TNF, IL-1β, and IL-8 in macrophages through MAPKs, NF-κB, and AP-1 signaling pathways. Furthermore, differential expression and release of JHP0290, HP1286, and HP1173 homologues was observed among H. pylori strains (papers II, III, IV). 

    Due to their ability to regulate multiple host cell responses, proteins JHP0290, HP1286, and HP1173 could play an important role in bacterial pathogenesis.

     

  • 29.
    Garcia-Bennett, Alfonso E.
    et al.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Konig, Niclas
    Abrahamsson, Ninnie
    Kozhevnikova, Mariya
    Zhou, Chunfang
    Trolle, Carl
    Pankratova, Stanislava
    Berezin, Vladimir
    Kozlova, Elena N.
    In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles2014In: Nanomedicine, ISSN 1743-5889, E-ISSN 1748-6963, Vol. 9, no 16, p. 2457-2466Article in journal (Refereed)
    Abstract [en]

    Aim: Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cells into MN precursors involves factors and their repeated administration. We investigated if delivery of factors loaded into mesoporous nanoparticles could be effective for stem cell differentiation in vitro. Materials & methods: We used a mouse embryonic stem cell line expressing green fluorescent protein under the promoter for the MN-specific gene Hb9 to visualize the level of MN differentiation. The differentiation of stem cells was evaluated by expression of MN-specific transcription factors monitored by quantitative real-time PCR reactions and immunocytochemistry. Results: Mesoporous nanoparticles have strong affiliation to the embryoid bodies, penetrate inside the embryoid bodies and come in contact with differentiating cells. Conclusion: Repeated administration of soluble factors into a culture medium can be avoided due to a sustained release effect using mesoporous silica.

  • 30.
    Gilderson, Gwen
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Transmembrane proton transfer in biological systems: an investigation of bacterial and archaeal haem-copper oxidases2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Haem-copper oxidases are membrane-bound protein complexes that catalyse the reduction of dioxygen to water in the respiratory chain. These enzyme complexes are molecular machines that couple the free energy released from this reaction to pump protons across the membrane. Pumped protons as well as protons used for dioxygen reduction to water are taken up from the inside of the membrane, which contributes to maintaining an electrochemical gradient across the membrane. This gradient is used for other energy-requiring processes in the cell, among them the formation of ATP.

    This thesis focuses on the proton coupled reactions and the timing of the proton pumping events.

    Specific pathways provide rapid transfer of protons through haem-copper oxidases. Since the intermediates formed during reduction of dioxygen must rapidly and continuously be supplied with protons, the proton uptake from the outside bulk solution must be maintained at a high rate. One part of this thesis concerns the effect of altering the surrounding of the entrance of one of the proton pathways on the proton-uptake rates.

    The directionality of proton transfer through the membrane must be controlled by the enzyme to prevent the dissipation of the electrochemical gradient. A specific pump element within one of the proton pathways has been proposed to control this directionality. A part of this thesis addresses the proposed pump element.

    The timing of proton pumping, monitored using a pH sensitive dye present in the outside bulk solution, was investigated in oxidase incorporated into closed artificial membranes. The results show that proton release occurs on a millisecond-time scale. Since the results from other studies were interpreted to indicate also faster proton pumping events, it is speculated that the membrane surface plays an important role in the delay of the proton release to the bulk solution.

  • 31.
    Glaser, Elzbieta
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bhushan, Shashi
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Two novel mitochondrial and chloroplastic targeting-peptide-degrading peptidasomes in A. thaliana, AtPreP1 and AtPreP2.2006In: Biol Chem, ISSN 1431-6730, Vol. 387, no 10-11, p. 1441-7Article in journal (Refereed)
    Abstract [en]

    Two novel metalloendopeptidases in Arabidopsis thaliana, AtPreP1 and AtPreP2, are responsible for the degradation of targeting peptides in mitochondria and chloroplasts. Both AtPreP1 and AtPreP2 contain ambiguous targeting peptides and are dually targeted to both organelles. The proteases also have the capacity to degrade unstructured peptides of up to 65 amino acid residues, but not small proteins. The catalysis occurs in a huge catalytic chamber revealed by the crystal structure of AtPreP1 at 2.1 A. The enzymes show a preference for basic and small uncharged amino acids or serines at the cleavage sites. Despite similarities in cleavage specificities, cleavage-site recognition differs for both proteases and is context- and structure-dependent. The AtPreP1 and AtPreP2 genes are differentially expressed in Arabidopsis.

  • 32.
    Gurmu, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural Studies of Microbial Proteins - From Escherichia coli and Herpesviruses2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Structure biology concerns the study of the molecular structures of biological macromolecules, such as proteins, and how these relate to the function. Protein structures are also of importance in structure-based drug design. In this thesis, the work has been carried out in two different projects. The first project concerns structural studies of proteins from the bacterium Escherichia coli and the second of proteins from five different herpesviruses.

     The E. coli project resulted in the structural characterization of three proteins: CaiB, RibD, and YhaK. CaiB is a type-III CoA transferase involved in the metabolism of carnitine. Its molecular structure revealed a spectacular fold where two monomers were interlaced forming an interlocked dimer. RibD, a bi-functional enzyme, catalyzes two consecutive reactions during riboflavin biosynthesis. In an attempt to characterize the mechanism of action of the N-terminal reductase domain, the structure of RibD was also determined in two binary complexes with the oxidized cofactor, NADP+, and with the substrate analogue ribose-5-phosphate. YhaK is a protein of unknown function normally found in low abundance in the cytosol of E. coli and was previously annotated to be a member of the Pirin family. However, some structural features seem to distinguish YhaK from these other Pirin proteins and we showed that YhaK might be regulated by reactive oxygen species.

     The Herpesvirus project resulted in the structural determination of two proteins, the SOX protein and ORF60 from Kaposi’s sarcoma associated herpesvirus (KSHV). SOX, a bi-functional shutoff and exonuclease protein, is involved in the maturation and packaging of the viral genome into the viral capsid and in the host shutoff of cellular proteins at the mRNA level. The SOX structure was also used for modeling DNA binding. The crystallization and preliminary structural studies of ORF60, the small R2 subunit of the ribonucleotide reductase (RNR) from KSHV is also discussed.

  • 33.
    Gyllberg, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Löfgren Söderberg, Kajsa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Almeida, Ana
    Bedecs, Katarina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Altered membrane distribution and increased Fyn activity in scrapie-infected neuronal cellsManuscript (preprint) (Other academic)
    Abstract [en]

    The suggested cause of Prion diseases is conversion of the cellular prion protein (PrPC) into aberrant scrapie prion protein (PrPSc) isoform triggered by the latter. PrPC is localized to membrane rafts, subdomains which are implicated in conversion. In this report we have analyzed the expression, plasma membrane distribution and activity of the raft associative Src family kinase member Fyn, in scrapie-infected neuronal cell lines. As a result of prion infection, the specific tyrosine kinase activity of Fyn is increased, although the protein level of Fyn kinase is reduced. This results in an increased overall tyrosine phosphorylation in the infected cells. Interestingly, prion infection also induced a membrane redistribution of Fyn from non-raft to raft-membrane subdomains, following the distribution of PrPSc, as shown by immunoblotting of flotation-fractions after density gradient centrifugation of Triton X-100 extracted cell extracts. This indicates a persistent Fyn activation, possibly due to clustering of intracellular Fyn kinases as a result of PrPSc association to membrane rafts. An increased Fyn kinase activity followed by a dramatic increase in tyrosine phosphorylation may cause and/or contribute to synaptic disorganization and loss which are fundamental features of prion disease.

  • 34.
    Haglund, Ellinor
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindberg, Magnus O.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Changes of protein folding pathways by circular permutation. Overlapping nuclei promote global cooperativity.2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 41, p. 27904-27915Article in journal (Refereed)
    Abstract [en]

    The evolved properties of proteins are not limited to structure and stability but also include their propensity to undergo local conformational changes. The latter, dynamic property is related to structural cooperativity and is controlled by the folding-energy landscape. Here we demonstrate that the structural cooperativity of the ribosomal protein S6 is optimized by geometric overlap of two competing folding nuclei: they both include the central beta-strand 1. In this way, folding of one nucleus catalyzes the formation of the other, contributing to make the folding transition more concerted overall. The experimental evidence is provided by an extended set of circular permutations of S6 that allows quantitative analysis of pathway plasticity at the level of individual side chains. Because similar overlap between competing nuclei also has been discerned in other proteins, we hypothesize that the coupling of several small nuclei into extended "supernuclei" represents a general principle for propagating folding cooperativity across large structural distances.

  • 35. Hansson Petersen, Camilla A.
    et al.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Behbahani, Homira
    Wiehager, Birgitta
    Pavlov, Pavel F
    Alafuzoff, Irina
    Leinonen, Ville
    Ito, Akira
    Winblad, Bengt
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ankarcrona, Maria
    The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 35, p. 13145-13150Article in journal (Refereed)
    Abstract [en]

    The amyloid beta-peptide (Abeta) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by Abeta and accumulation of Abeta has been detected in mitochondria. Because Abeta is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial Abeta uptake. Our results from rat mitochondria show that Abeta is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed Abeta association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of Abeta to mitochondrial cristae. A similar distribution pattern of Abeta in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for Abeta in mitochondria and demonstrate both in vitro and in vivo that Abeta is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied Abeta can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of Abeta, a peptide considered to be of major significance in Alzheimer's disease.

  • 36.
    Heddad, Mounia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Light stress proteins from Arabidopsis thaliana: identification, characterisation and expression2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Plants are major players in the vital process of photosynthesis that takes place in organelles called chloroplasts. Plant's immobility makes them victims of the tremendous fluctuations in light intensity and quality that often lead to inhibition of their physiological functions. In order to maintain the photosynthetic processes under light stress conditions, plants have developed several protective mechanisms. In this thesis, one such protective strategy operating in the chloroplasts, namely the accumulation of light stress proteins from the Elip (early light-induced proteins) family of Arabidopsis thaliana is presented. Elips in higher plants are nuclear-encoded proteins located in the thylakoid membranes and related to the chlorophyll a/b-binding proteins of photosystem I and II. A photoprotective function either by binding of released free chlorophylls and thus preventing the formation of free radicals or by thermal dissipation of excess energy was proposed for Elip family members. Using genomic resources available for A. thaliana, two members of the classical three-helix Elips, a new class of two-helix stress-enhanced proteins (called Sep) and a Elip-like one-helix protein (called Ohp 2) were identified and characterised at the molecular level. We demonstrated that both light and retrograde signalling are involved in the regulation of these proteins and that this regulation occurrs independently at transcript and protein levels. We showed that while Elips and Seps are located in photosystem II, Ohp 2 was found in photosystem I, thus providing evidence for a novel protective mechanism present in this photosystem. This work further demonstrates the involvement of chlorophyll a in the posttranslational regulation of Elip expression, thus supporting the proposed photoprotective function of these proteins. Finally, the distribution of the Elip family members through oxygenic photosynthetic organisms from the prokaryotic cyanobacteria, all throughout primitive algae and finally to higher plants was analysed and a novel model for the evolution of Elips and chlorophyll a/b-binding proteins in higher plants and green algae is proposed.

  • 37.
    Hedin, Linnea E.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bernsel, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hennerdal, Aron
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Illergård, Kristoffer
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Enquist, Karl
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kauko, Anni
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lerch-Bader, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 396, no 1, p. 221-229Article in journal (Refereed)
    Abstract [en]

    In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.

  • 38. Hedskog, Louise
    et al.
    Pinho, Catarina Moreira
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Filadi, Riccardo
    Rönnbäck, Annica
    Hertwig, Laura
    Wiehager, Birgitta
    Larssen, Pia
    Gellhaar, Sandra
    Sandebring, Anna
    Westerlund, Marie
    Graff, Caroline
    Winblad, Bengt
    Galter, Dagmar
    Behbahani, Homira
    Pizzo, Paola
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ankarcrona, Maria
    Modulation of the endoplasmic reticulum-mitochondria interface in Alzheimer's disease and related models2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 19, p. 7916-7921Article in journal (Refereed)
    Abstract [en]

    It is well-established that subcompartments of endoplasmic reticulum (ER) are in physical contact with the mitochondria. These lipid raft-like regions of ER are referred to as mitochondria-associated ER membranes (MAMs), and they play an important role in, for example, lipid synthesis, calcium homeostasis, and apoptotic signaling. Perturbation of MAM function has previously been suggested in Alzheimer's disease (AD) as shown in fibroblasts from AD patients and a neuroblastoma cell line containing familial presenilin-2 AD mutation. The effect of AD pathogenesis on the ER-mitochondria interplay in the brain has so far remained unknown. Here, we studied ER-mitochondria contacts in human AD brain and related AD mouse and neuronal cell models. We found uniform distribution of MAM in neurons. Phosphofurin acidic cluster sorting protein-2 and sigma 1 receptor, two MAM-associated proteins, were shown to be essential for neuronal survival, because siRNA knockdown resulted in degeneration. Up-regulated MAM-associated proteins were found in the AD brain and amyloid precursor protein (APP)(Swe/Lon) mouse model, in which up-regulation was observed before the appearance of plaques. By studying an ER-mitochondria bridging complex, inositol-1,4,5-triphosphate receptor-voltage-dependent anion channel, we revealed that nanomolar concentrations of amyloid beta-peptide increased inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevated the number of ER-mitochondria contact points and mitochondrial calcium concentrations. Our data suggest an important role of ER-mitochondria contacts and cross-talk in AD pathology.

  • 39.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Radical generation and stabilisation in ribonucleotide reductase R22003Doctoral thesis, monograph (Other academic)
    Abstract [en]

    Diiron carboxylate proteins contain a cofactor that consists of two iron atoms coordinated by carboxylate and histidine ligands. These proteins perform a multitude of chemical reactions in the cell that generally involve activation of molecular oxygen at the diiron site.

    Ribonucleotide reductase is the only enzyme that performs de novo synthesis of all four deoxyribonucleotides, the building blocks of DNA. The R2 protein of Class-I ribonucleotide reductase, which is a diiron carboxylate protein, utilises the high-valent iron-oxygen species generated at the diiron site to produce a stable tyrosyl radical required for enzymatic activity.

    In this work, X-ray crystallography and EPR are used to study the R2 protein from Escherichia coli with the goal of understanding its mechanism of oxygen activation, radical generation and radical stabilisation. Based on these studies a detailed structural mechanism is proposed, which might be common to several oxygen activating diiron carboxylate proteins. The orientation of the active radical species in R2 has also been determined. In addition, these results provide a rationale for the unusual stability of the radical.

    Crystal structures of R2 proteins from two other species, Corynebacterium ammoniagenes and Chlamydia trachomatis, have also been solved.

    The C. ammoniagenes protein has been reported to be manganese-dependent. The results presented here, however, support dependence of iron and not manganese.

    Sequence alignments indicate that the chlamydial R2s lack the, otherwise conserved, radical harbouring tyrosine. This is confirmed by the structure, which also reveals other unique features in the diiron site. Hypotheses regarding the function of the protein and the reason for the differences are presented.

  • 40.
    Höhna, Sebastian
    Stockholm University, Faculty of Science, Department of Mathematics.
    Fast simulation of reconstructed phylogenies under global time-dependent birth-death processes2013In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 29, no 11, p. 1367-1374Article in journal (Refereed)
    Abstract [en]

    Motivation: Diversification rates and patterns may be inferred from reconstructed phylogenies. Both the time-dependent and the diversity-dependent birthdeath process can produce the same observed patterns of diversity over time. To develop and test new models describing the macro-evolutionary process of diversification, generic and fast algorithms to simulate under these models are necessary. Simulations are not only important for testing and developing models but play an influential role in the assessment of model fit.

    Results: In the present article, I consider as the model a global time-dependent birthdeath process where each species has the same rates but rates may vary over time. For this model, I derive the likelihood of the speciation times from a reconstructed phylogenetic tree and show that each speciation event is independent and identically distributed. This fact can be used to simulate efficiently reconstructed phylogenetic trees when conditioning on the number of species, the time of the process or both. I show the usability of the simulation by approximating the posterior predictive distribution of a birthdeath process with decreasing diversification rates applied on a published bird phylogeny (family Cettiidae).

    Availability: The methods described in this manuscript are implemented in the R package TESS, available from the repository CRAN (http://cran.r-project.org/web/packages/TESS/).

  • 41. Kacprzak, Justyna
    et al.
    Kuszewski, Tomasz
    Lankoff, Anna
    Mueller, Wolfgang-Ulrich
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lisowska, Halina
    Individual variations in the micronucleus assay for biological dosimetry after high dose exposure2013In: Mutation research. Genetic toxicology and environmental mutagenesis, ISSN 1383-5718, E-ISSN 1879-3592, Vol. 756, no 1-2, p. 196-200Article in journal (Refereed)
    Abstract [en]

    The micronucleus assay is widely used as a biological dosimeter. Due to an inhibitory effect of radiation on cell proliferation the assay yields satisfactory results only when the absorbed dose is below about 5 Gy. In 2002 Muller and Rode suggested that a modified version of the test, based on the analysis of the ratio of trinucleated to tetranucleated cells and the frequency of micronuclei (Mn) in binucleated cells containing at least one Mn, can be applied to detect a dose reaching 15 Gy (Mutat. Res. 502 (2002) 47-51). Their conclusion was based on the results of experiments with lymphocytes from one donor and nothing is known about the possible influence of individual variability on the applicability of the Mn test to detect high doses of radiation. The aim of the present study was to validate the modified micronucleus assay with lymphocytes of 5 donors. Their blood was exposed to 0, 5, 10, 15 and 20 Gy of Co-60 gamma rays. The levels of Mn and of cell proliferation were assessed using various approaches. A strong inter-individual variability was observed for all endpoints. The results clearly show that the assessment of cell proliferation is essential for the interpretation of results. Unfortunately, it was not possible to identify one single proliferation marker that gives all necessary information.

  • 42. Karlsson, Oskar
    et al.
    Jiang, Liying
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Ersson, Lisa
    Malmström, Tim
    Ilag, Leopold L.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Brittebo, Eva B.
    Environmental neurotoxin interaction with proteins: Dose-dependent increase of free and protein-associated BMAA (beta-N-methylamino-L-alanine) in neonatal rat brain2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 15570Article in journal (Refereed)
    Abstract [en]

    beta-Methylamino-L-alanine (BMAA) is implicated in the aetiology of neurodegenerative disorders. Neonatal exposure to BMAA induces cognitive impairments and progressive neurodegenerative changes including intracellular fibril formation in the hippocampus of adult rats. It is unclear why the neonatal hippocampus is especially vulnerable and the critical cellular perturbations preceding BMAA-induced toxicity remains to be elucidated. The aim of this study was to compare the level of free and protein-associated BMAA in neonatal rat brain and peripheral tissues after different exposures to BMAA. Ultra-high performance liquid chromatography-tandem mass spectrometry analysis revealed that BMAA passed the neonatal blood-brain barrier and was distributed to all studied brain areas. BMAA was also associated to proteins in the brain, especially in the hippocampus. The level in the brain was, however, considerably lower compared to the liver that is not a target organ for BMAA. In contrast to the liver there was a significantly increased level of protein-association of BMAA in the hippocampus and other brain areas following repeated administration suggesting that the degradation of BMAA-associated proteins may be lower in neonatal brain than in the liver. Additional evidence is needed in support of a role for protein misincorporation in the neonatal hippocampus for long-term effects of BMAA.

  • 43.
    Kauko, Anni
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hedin, Linnea E
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Thebaud, Estelle
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Repositioning of transmembrane alpha-helices during membrane protein folding2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, no 1, p. 190-201Article in journal (Refereed)
    Abstract [en]

    We have determined the optimal placement of individual transmembrane helices in the Pyrococcus horikoshii Glt(Ph) glutamate transporter homolog in the membrane. The results are in close agreement with theoretical predictions based on hydrophobicity, but do not, in general, match the known three-dimensional structure, suggesting that transmembrane helices can be repositioned relative to the membrane during folding and oligomerization. Theoretical analysis of a database of membrane protein structures provides additional support for this idea. These observations raise new challenges for the structure prediction of membrane proteins and suggest that the classical two-stage model often used to describe membrane protein folding needs to be modified.

  • 44. Kilk, Kalle
    et al.
    Mahlapuu, Riina
    Soomets, Ursel
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Analysis of in vitro toxicity of five cell-penetrating peptides by metabolic profiling2009In: Toxicology, ISSN 0300-483X, E-ISSN 1879-3185, Vol. 265, no 3, p. 87-95Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are promising candidates for safe and efficient delivery vectors for a wide range of cargoes. However, any compound that is internalized into cells may affect the cell homeostasis. The plethora of possible biological responses makes large scale “omics” studies appealing approaches for hunting any unsuspected side-effects and evaluate the toxicity of drug candidates. Here we have compared the alterations in cytosolic metabolome of CHO cells caused by five representatives of the most common CPPs: transportan (TP), penetratin (pAntp), HIV Tat derived peptide (pTat), nonaarginine (R9) and model amphipathic peptide (MAP). Analysis was done by liquid chromatography–mass spectrometry techniques, principal component analysis and heatmap displays. Results showed that the intracellular metabolome was the most affected by TP followed by pTat and MAP. Only minor changes could be associated with pAntp or R9 treatment. The cells could recover from a treatment with 5 μM TP, but no recovery was observed at higher concentration. Both metabolomic and control experiments showed that TP affected cellular redox potential, depleted energy and the pools of purines and pyrimidines. In conclusion, we have performed a metabolomic analysis comparing the safety of cell-penetrating peptides and demonstrate the toxicity of one of them.

  • 45. Krõlov, Katrin
    et al.
    Uusna, Julia
    Grellier, Tiia
    Andresen, Liis
    Jevtuševskaja, Jekaterina
    Tulp, Indrek
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Implementation of antimicrobial peptides for sample preparation prior to nucleic acid amplification in point-of-care settings2017In: Expert Review of Molecular Diagnostics, ISSN 1473-7159, E-ISSN 1744-8352, Vol. 17, no 12, p. 1117-1125Article in journal (Refereed)
    Abstract [en]

    Background: A variety of sample preparation techniques are used prior to nucleic acid amplification. However, their efficiency is not always sufficient and nucleic acid purification remains the preferred method for template preparation. Purification is difficult and costly to apply in point-of-care (POC) settings and there is a strong need for more robust, rapid, and efficient biological sample preparation techniques in molecular diagnostics. Methods: Here, the authors applied antimicrobial peptides (AMPs) for urine sample preparation prior to isothermal loop-mediated amplification (LAMP). AMPs bind to many microorganisms such as bacteria, fungi, protozoa and viruses causing disruption of their membrane integrity and facilitate nucleic acid release. Results: The authors show that incubation of E. coli with antimicrobial peptide cecropin P1 for 5 min had a significant effect on the availability of template DNA compared with untreated or even heat treated samples resulting in up to six times increase of the amplification efficiency. Conclusion: These results show that AMPs treatment is a very efficient sample preparation technique that is suitable for application prior to nucleic acid amplification directly within biological samples. Furthermore, the entire process of AMPs treatment was performed at room temperature for 5 min thereby making it a good candidate for use in POC applications.

  • 46. Kurrikoff, Kaido
    et al.
    Gestin, Maxime
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Recent in vivo advances in cell-penetrating peptide-assisted drug delivery2016In: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 13, no 3, p. 373-387Article, review/survey (Refereed)
    Abstract [en]

    Introduction: Delivery of macromolecular drugs is an important field in medical research. However, macromolecules are usually unable to cross the cell membrane without the assistance of a delivery system. Cell penetrating peptides (CPPs) are unique tools to gain access to the cell interior and deliver a bioactive cargo into the cytosol or nucleus. In addition to macromolecular delivery, CPPs have been used to deliver smaller bioactive molecules. Therefore CPPs have become an intensive field of research for medical treatment.

    Areas covered: In this review, we highlight studies that include CPP in vivo disease models. We review different strategies and approaches that have been used, with specific attention on recent publications. The approaches that have been used include CPP–cargo covalent conjugation strategies and nanoparticle strategies. Various additional strategies have been used to achieve disease targeting, including active targeting, passive targeting, and combined active/passive strategies. As a result, delivery of various types of molecule has been achieved, including small drug molecules, proteins and nucleic acid-based macromolecules (e.g. siRNA, antisense nucleotides and plasmid DNA).

    Expert Opinion: Despite recent advances in the field, confusions surrounding CPP internalization mechanisms and intracellular trafficking are hindering the development of new and more efficient vectors. Nevertheless, the recent increase in the number of publications containing in vivo CPP utilization looks promising that the number of clinical trials would also increase in the near future.

  • 47.
    Lagerstedt, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reeve, Ian
    Voss, John C
    Persson, Bengt L
    The structure and function of GTP binding protein Gtr1 and its role in phosphate transport in Saccharomyces cerevisiae2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 2, p. 511-517Article in journal (Refereed)
    Abstract [en]

    The Pho84 high-affinity phosphate permease is the primary phosphate transporter in the yeast Saccharomyces cerevisiae under phosphate-limiting conditions. The soluble G protein, Gtr1, has previously been suggested to be involved in the derepressible Pho84 phosphate uptake function. This idea was based on a displayed deletion phenotype of Δgtr1 similar to the Δpho84 phenotype. As of yet, the mode of interaction has not been described. The consequences of a deletion of gtr1 on in vivo Pho84 expression, trafficking and activity, and extracellular phosphatase activity were analyzed in strains synthesizing either Pho84−green fluorescent protein or Pho84−myc chimeras. The studies revealed a delayed response in Pho84-mediated phosphate uptake and extracellular phosphatase activity under phosphate-limiting conditions. EPR spectroscopic studies verified that the N-terminal G binding domain (residues 1−185) harbors the nucleotide responsive elements. In contrast, the spectra obtained for the C-terminal part (residues 186−310) displayed no evidence of conformational changes upon GTP addition.

  • 48.
    Lang, Lisa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zetterström, Per
    Brännström, Thomas
    Marklund, Stefan L.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    SOD1 aggregation in ALS mice shows simplistic test tube behavior2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 32, p. 9878-9883Article in journal (Refereed)
    Abstract [en]

    A longstanding challenge in studies of neurodegenerative disease has been that the pathologic protein aggregates in live tissue are not amenable to structural and kinetic analysis by conventional methods. The situation is put in focus by the current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quantitative in vivo comparison. In this study, we bridge this gap by presenting a direct comparison of the aggregation kinetics of the ALS-associated protein superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quantitative antibody assays show that the SOD1 fibrillation kinetics in vitro mirror with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggests that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathology and organism-level manifestation of protein aggregation phenomena in general.

  • 49.
    Larsson, Karl-Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Allosteric Regulation and Radical Transfer in Ribonucleotide Reductase2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The stability of biological life over time requires that the integrity of the genetic material of an organism, the genome, be maintained as it is passed on from one generation to the next. All known cellular life has DNA-based genomes, which are duplicated before each cell division in a process called replication. During and after DNA replication, the integrity of the genetic information is maintained by various proofreading and DNA repair mechanisms. The accuracy of DNA replication and repair is affected by DNA precursor pool imbalances. Feedback regulation of the enzymes involved in DNA precursor biosynthesis has evolved in parallel with the DNA replication and repair system in order to ensure stable precursor pools.

    Ribonucleotide reductase (RNR), an enzyme that irreversibly reduces ribonucleotides into deoxyribonucleotides, is a key component in the regulation of the DNA precursor pools. It has sophisticated allosteric regulatory mechanisms that govern both overall activity and substrate specificity, responding to the cellular concentrations of ATP and of the triphosphate forms of the product deoxyribonucleotides, the final DNA precursors.

    Using X-ray crystallography we have solved several structures of two ribonucleotide reductases, an anaerobic (class III) enzyme from Bacteriophage T4 and a coenzyme B12-dependent (class II) enzyme from Thermotoga maritima, in complex with various nucleotides and cofactors. The structural information reveals a complete molecular mechanism for the allosteric substrate specificity regulation of class II RNRs which, due to structural homology, is likely also to be valid for the aerobic class I RNRs. The work on the class III RNRs has produced a partial mechanism for the specificity regulation of this class. Both mechanisms utilize Loop 2, a conserved structural element, in the transmission of the allosteric signal.

    The discovery of a metal binding domain in the anaerobic RNRs and details of coenzyme B12 binding shed more light on generation and transfer of the protein based radicals used in the reduction reaction.

  • 50.
    Larsson, Veronica J.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    The roles of inner nuclear membrane proteins during interphase and mitosis2014Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The nuclear envelope (NE) consists of two concentric membranes, the outer nuclear membrane (ONM) and the inner nuclear membrane (INM). The LINC (linker of nucleoskeleton and cytoskeleton) complex spans both the ONM and the INM connecting the cytoskeleton to the nucleoskeleton and chromatin. Only a few of the known INM proteins have been functionally characterized and shown to have important roles in chromatin organisation. Defects in the genes coding for proteins in the INM and the nuclear lamina give rise to serious human diseases, called envelopathies.

    In 2009 (Buch et al. 2009) our group made two major discoveries. We showed for the first time, that an integral INM protein distributed along the microtubules of the mitotic spindle. This protein was therefore named Samp1, Spindle Associated Membrane Protein 1. The second discovery was that depletion of Samp1 caused detachment of the centrosome from the NE, suggesting that Samp1 is associated with the microtubule cytoskeleton both in interphase and mitosis.

    In this thesis we continued to investigate Samp1´s role during interphase. We also wanted to investigate the localisation of Samp1 in the mitotic spindle and possible function during mitosis. We show that the expression of Samp1 mutants and depletion of Samp1 affects the distribution and organisation of A-type lamins, the LINC complex protein Sun1 and the LINC complex associated protein emerin. Thus, in interphase Samp1 is functionally connected to the LINC complex and the A-type lamina network. The LINC complex can help explain how the centrosomes detach from the NE in Samp1 depleted cells. In mitotic cells, we found that depletion of Samp1 caused prolonged metaphase and aberrant mitotic phenotypes such as bi-nucleation, enlarged nuclei and micronuclei. We also showed that Samp1 interacts with RanGTPase and importin-β, which are key players in assembling the mitotic spindle. Samp1 also modulates the levels of importin-β and NuMA in the mitotic spindle, which could explain the mitotic phenotypes that we se in Samp1 depleted cells. Here we present evidence showing, for the first time, that an INM protein is present on kinetochore microtubules and have an essential role for correct chromosome segregation and spindle assembly.

12 1 - 50 of 99
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf