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  • 51.
    Attoff, Kristina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Östlund Farrants, Ann-Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Johansson, Ylva
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cediel Ulloa, Andrea
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Acrylamide alters CREB and retinoic acid signaling pathways during differentiation of the human neuroblastoma SH-SY5Y cell lineManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Acrylamide is a known neurotoxic compound that we get exposed to through food and through the environment. It can cross the placental barrier as well as the blood-brain barrier resulting in exposure of the fetus and the infant child. We used the human neuroblastoma cell line SH-SY5Y to study the effects of non-cytotoxic acrylamide exposure during 9 days of differentiation on two differentially important signaling pathways, i.e. the retinoic acid receptor (RAR) and cAMP response element-binding protein (CREB) signaling in neurons. Our results showed that exposure of non-cytotoxic concentrations of acrylamide during 9 days of differentiation induced altered expression of multiple genes that are part of the CREB and RAR activation pathways, e.g. cellular retinoic acid binding protein 1, retinol binding protein 7, CREB5 and fibroblast growth factor receptor 2. Other well-established neuronal markers such as brain-derived neurotrophic factor, syntaxin binding protein 2, transforming growth factor beta 1, the dopaminergic markers monoamine oxidase A and dopamine receptor D2 as wells as the cholinergic marker choline O-acetyltransferase were also significantly altered by acrylamide. Our results reveal that acrylamide interferes with crucial pathways involved in neuronal differentiation in vitro and raise concerns over the potential toxic outcomes in humans.

  • 52. Azimzadeh, Omid
    et al.
    Scherthan, Harry
    Yentrapalli, Ramesh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Barjaktarovic, Zarko
    Ueffing, Marius
    Conrad, Marcus
    Neff, Frauke
    Calzada-Wack, Julia
    Aubele, Michaela
    Buske, Christian
    Atkinson, Michael J.
    Hauck, Stefanie M.
    Tapio, Soile
    Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediatemitochondrial impairment after ionising radiation2012Ingår i: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, nr 8, s. 2384-2395Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions. As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p <= 0.05) in irradiated hearts 24 h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives.

  • 53. Azimzadeh, Omid
    et al.
    Sievert, Wolfgang
    Sarioglu, Hakan
    Yentrapalli, ramesh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Barjaktarovic, Zarko
    Sriharshan, Arundhathi
    Ueffing, Marius
    Janik, Dirk
    Aichler, Michaela
    Atkinson, Michael J
    Multhoff, Gabriele
    Tapio, Soile
    PPAR Alpha: A Novel Radiation Target in Locally Exposed Mus musculus Heart Revealed by Quantitative Proteomics2013Ingår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, nr 6, s. 2700-2714Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Radiation exposure of the thorax is associated with a markedly increased risk of cardiac morbidity and mortality with a latency period of decades. Although many studies have confirmed the damaging effect of ionizing radiation on the myocardium and cardiac endothelial structure and function, the molecular mechanism behind this damage is not yet elucidated. Peroxisome proliferator-activated receptor alpha (PPAR alpha), a transcriptional regulator of lipid metabolism in heart tissue, has recently received great attention in the development of cardiovascular disease. The goal of this study was to investigate radiation-induced cardiac damage in general and the role of PPAR alpha in this process in particular. C57BL/6 mice received local heart irradiation with X-ray doses of 8 and 16 gray (Gy) at the age of 8 weeks. The mice were sacrificed 16 weeks later. Radiation-induced changes in the cardiac proteome were quantified using the Isotope Coded Protein Label (ICPL) method followed by mass spectrometry and software analysis. Significant alterations were observed in proteins involved in lipid metabolism and oxidative phosphorylation. Ionizing radiation markedly changed the phosphorylation and ubiquitination status of PPAR alpha. This was reflected as decreased expression of its target genes involved in energy metabolism and mitochondrial respiratory chain confirming the proteomics data. This study suggests that persistent alteration of cardiac metabolism due to impaired PPAR alpha activity contributes to the heart pathology after radiation.

  • 54. Bach, Dominik R.
    et al.
    Guitart-Masip, Marc
    Stockholms universitet, Samhällsvetenskapliga fakulteten, Centrum för forskning om äldre och åldrande (ARC), (tills m KI).
    Packard, Pau A.
    Miro, Julia
    Falip, Merce
    Fuentemilla, Lluis
    Dolan, Raymond J.
    Human Hippocampus Arbitrates Approach-Avoidance Conflict2014Ingår i: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 24, nr 5, s. 541-547Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Animal models of human anxiety often invoke a conflict between approach and avoidance [1, 2]. In these, a key behavioral assay comprises passive avoidance of potential threat and inhibition, both thought to be controlled by ventral hippocampus [2-6]. Efforts to translate these approaches to clinical contexts [7, 8] are hampered by the fact that it is not known whether humans manifest analogous approach-avoidance dispositions and, if so, whether they share a homologous neurobiological substrate [9]. Here, we developed a paradigm to investigate the role of human hippocampus in arbitrating an approach-avoidance conflict under varying levels of potential threat. Across four experiments, subjects showed analogous behavior by adapting both passive avoidance behavior and behavioral inhibition to threat level. Using functional magnetic resonance imaging (fMRI), we observe that threat level engages the anterior hippocampus, the human homolog of rodent ventral hippocampus [10]. Testing patients with selective hippocampal lesions, we demonstrate a causal role for the hippocampus with patients showing reduced passive avoidance behavior and inhibition across all threat levels. Our data provide the first human assay for approach-avoidance conflict akin to that of animal anxiety models. The findings bridge rodent and human research on passive avoidance and behavioral inhibition and furnish a framework for addressing the neuronal underpinnings of human anxiety disorders, where our data indicate a major role for the hippocampus.

  • 55.
    Baez, Sofia
    Stockholms universitet.
    Dopachrome and aminochrome, the oxidized metabolites of dopa and dopamine: studies on the molecular mechanisms underlying their reduction and conjugation with glutathione1999Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Dopamine, like other catecholamines can be oxidized to the corresponding ortho-quinone, which undergoes cyclization of its amino chain to cyclized dopamine ortho-quinone (aminochrome). Both o-quinones are toxic metabolites that may contribute to neurotoxic effects of these catecholamines. The oxidation of dopamine to o-quinone is believed to be one of the causes of the neurodegenerative process of Parkinson's disease.

    Although it is in generally accepted that free radicals are involved in the neurodegenerative process occurring in the dopaminergic neuron system in Parkinson's disease, the exact mechanism of neurodegeneration in vivo is still unknown. However, one possible source of free radicals in the dopaminergic neuron system in the central nerve system may involve the oxidation of dopamine to the corresponding o-quinone.

    L-Dopa is commonly used in the clinical management of Parkinson's disease. However, this treatment becomes gradually ineffective, due either to loss of efficacy or to appearance of side-effects, probably produced by the oxidative injury generated by the oxidation of dopa or of its metabolites.

    We report in this study that the oxidation of dopa and dopamine to the corresponding o-quinone and the followed cyclization, at physiological pH, is not itself responsible for the formation of reactive oxygen species. In addition, we show that the reduction of cyclized o-quinones of dopamine and the subsequent autoxidation is the step in which reactive species are formed.

    Formation of reactive oxygen species during one- or two-electron reduction of aminochrome and dopachrome has been studied in vitro by using NADPH-cytochrome P450 reductase and DT-diaphorase, respectively. The results suggest that DT-diaphorase, SOD, catalase (glutathione peroxidase) and sulfotransferase constitute the cellular defenses against formation of reactive oxygen species during reduction of aminochrome and dopachrome.

    We also studied the ability of human glutathione transferases to conjugate cyclized catechol o-quinones such as aminochrome and dopachrome and found that GSTs, in particularly GST M2-2 catalyzes GSH conjugation of dopachrome and aminochrome, preventing the formation of ROS and reactive catechol metabolites. The neuroprotective role of GST M2-2 suggested in the present study is depend on the presence of the enzyme in relevants regions of the human brain, regions where cell damage is observed in diseases such as schizoprenia and Parkinson's disease.

  • 56.
    Bakali, Amin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structural studies of three cell signaling proteins: crystal structures of EphB1, PTPA, and YegS2007Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases.

    The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A.

    Finally, the structure of YegS, an Escherichia coli protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.

  • 57.
    Baltscheffsky, Herrick
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Persson, Bengt
    On an Early Gene for Membrane-Integral Inorganic Pyrophosphatase in the Genome of an Apparently Pre-LUCA Extremophile, the Archaeon Candidatus Korarchaeum cryptofilum2014Ingår i: Journal of Molecular Evolution, ISSN 0022-2844, E-ISSN 1432-1432, Vol. 78, nr 2, s. 140-147Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A gene for membrane-integral inorganic pyrophosphatase (miPPase) was found in the composite genome of the extremophile archaeon Candidatus Korarchaeum cryptofilum (CKc). This korarchaeal genome shows unusual partial similarity to both major archaeal phyla Crenarchaeota and Euryarchaeota. Thus this Korarchaeote might have retained features that represent an ancestral archaeal form, existing before the occurrence of the evolutionary bifurcation into Crenarchaeota and Euryarchaeota. In addition, CKc lacks five genes that are common to early genomes at the LUCA border. These two properties independently suggest a pre-LUCA evolutionary position of this extremophile. Our finding of the miPPase gene in the CKc genome points to a role for the enzyme in the energy conversion of this very early archaeon. The structural features of its miPPase indicate that it can pump protons through membranes. An miPPase from the extremophile bacterium Caldicellulosiruptor saccharolyticus also has a sequence indicating a proton pump. Recent analysis of the three-dimensional structure of the miPPase from Vigna radiata has resulted in the recognition of a strongly acidic substrate (orthophosphate: Pi, pyrophosphate: PPi) binding pocket, containing 11 Asp and one Glu residues. Asp (aspartic acid) is an evolutionarily very early proteinaceous amino acid as compared to the later appearing Glu (glutamic acid). All the Asp residues are conserved in the miPPase of CKc, V. radiata and other miPPases. The high proportion of Asp, as compared to Glu, seems to strengthen our argument that biological energy conversion with binding and activities of orthophosphate (Pi) and energy-rich pyrophosphate (PPi) in connection with the origin and early evolution of life may have started with similar or even more primitive acidic peptide funnels and/or pockets.

  • 58. Banci, Lucia
    et al.
    Blazevits, Olga
    Cantini, Francesca
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lang, Lisa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Luchinat, Claudio
    Mao, Jiafei
    Oliveberg, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ravera, Enrico
    Solid-state NMR studies of metal-free SOD1 fibrillar structures2014Ingår i: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, nr 4-5, s. 659-666Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Copper-zinc superoxide dismutase 1 (SOD1) is present in the protein aggregates deposited in motor neurons of amyotrophic lateral sclerosis (ALS) patients. ALS is a neurodegenerative disease that can be either sporadic (ca. 90 %) or familial (fALS). The most widely studied forms of fALS are caused by mutations in the sequence of SOD1. Ex mortuo SOD1 aggregates are usually found to be amorphous. In vitro SOD1, in its immature reduced and apo state, forms fibrillar aggregates. Previous literature data have suggested that a monomeric SOD1 construct, lacking loops IV and VII, (apoSOD Delta IV-VII), shares the same fibrillization properties of apoSOD1, both proteins having the common structural feature of the central beta-barrel. In this work, we show that structural information can be obtained at a site-specific level from solid-state NMR. The residues that are sequentially assignable are found to be located at the putative nucleation site for fibrillar species formation in apoSOD, as detected by other experimental techniques.

  • 59. Bano-Polo, Manuel
    et al.
    Martinez-Gill, Luis
    Wallner, Björn
    Nieva, Jose L.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Mingarro, Ismael
    Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion2013Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, nr 4, s. 830-840Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    alpha-Helical hairpins, consisting of a pair of closely spaced transmembrane (TM) helices that are connected by a short interfacial turn, are the simplest structural motifs found in multi-spanning membrane proteins. In naturally occurring hairpins, the presence of polar residues is common and predicted to complicate membrane insertion. We postulate that the pre-packing process offsets any energetic cost of allocating polar and charged residues within the hydrophobic environment of biological membranes. Consistent with this idea, we provide here experimental evidence demonstrating that helical hairpin insertion into biological membranes can be driven by electrostatic interactions between closely separated, poorly hydrophobic sequences. Additionally, we observe that the integral hairpin can be stabilized by a short loop heavily populated by turn-promoting residues. We conclude that the combined effect of TM-TM electrostatic interactions and tight turns plays an important role in generating the functional architecture of membrane proteins and propose that helical hairpin motifs can be acquired within the context of the Sec61 translocon at the early stages of membrane protein biosynthesis. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains from primary structures.

  • 60. Barregard, Lars
    et al.
    Haghdoost, Siamak
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Cooke, Marcus S.
    Human and Methodological Sources of Variability in the Measurement of Urinary 8-Oxo-7,8-dihydro-2 '-deoxyguanosine2013Ingår i: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 18, nr 18, s. 2377-2391Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aims: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (r(p) 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended.

  • 61.
    Barth, Andreas
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Haris, Parvez I.De Montfort University, Leicester, UK.
    Biological and Biomedical Infrared Spectroscopy2009Samlingsverk (redaktörskap) (Övrigt vetenskapligt)
  • 62. Bassett, Andrew R.
    et al.
    Azzam, Ghows
    Wheatley, Lucy
    Tibbit, Charlotte
    Rajakumar, Timothy
    McGowan, Simon
    Stanger, Nathan
    Ewels, Philip Andrew
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Taylor, Stephen
    Ponting, Chris P.
    Liu, Ji-Long
    Sauka-Spengler, Tatjana
    Fulga, Tudor A.
    Understanding functional miRNA-target interactions in vivo by site-specific genome engineering2014Ingår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, s. 4640-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    MicroRNA (miRNA) target recognition is largely dictated by short 'seed' sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA-target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA-MRE interactions at any point during development.

  • 63.
    Baumgarten, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Protein production in the E. coli cell envelope2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Proteins fulfil essential functions in every cell and malfunctioning proteins are often the cause of diseases. On the other hand, proteins like antibody fragments or hormones can be used to treat diseases. Proteins are often produced in the bacterium Escherichia coli so that they can be studied to understand their (mal)function or so that they can be used to treat a disease. Unfortunately, producing proteins in the cell envelope of E. coli, like integral membrane proteins, which are important drug targets, and secretory proteins like antibody fragments and hormones, often results in unsatisfactory yields. Therefore, the objectives of this doctoral thesis were to identify bottlenecks that can limit the production of recombinant proteins in the cell envelope of E. coli and to try to overcome these bottlenecks. In the first study, we isolated and characterized the E. coli membrane protein production strain Mt56(DE3). This strain, in which the target gene expression intensity is strongly reduced, outcompetes the standard E. coli membrane protein production strains for most targets tested. In the second and third study we focused on the production of secretory proteins, i.e., proteins that are translocated across the inner membrane into the periplasm of E. coli. First, we investigated the impact of the targeting pathway used to direct a secretory protein to the translocation machinery on the cell physiology and protein production yields. We found that the co-translational targeting of a produced protein saturates the capacity of the translocation machinery resulting in heavily impaired biomass formation and low protein production yields. In contrast, post-translational targeting of a produced protein did not saturate the capacity of the protein translocation machinery resulting in hardly affected biomass formation and high protein production yields. In the third study we investigated how optimizing the production of a co-translationally targeted protein, by harmonizing its production rate with the capacity of the protein translocation machinery, affects the physiology of the cell. We found that, in stark contrast to the non-optimized condition, the optimized production did not affect the composition of the E. coli proteome. This surprising finding indicates that a protein can be produced efficiently in the periplasm of E. coli without compromising the physiology of the cell. In the last study we aimed at developing an outer membrane vesicle-based tuberculosis vaccine. To this end, an E. coli strain was created that produced outer membrane vesicles coated with different tuberculosis antigens. It was shown that a homogenous population of vesicles was produced, which will hopefully facilitate the isolation of these vesicles on an industrial scale.

  • 64. Bavec, Aljosa
    et al.
    Jiang, Yang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Zorko, Matjaz
    Role of cysteine 341 and arginine 348 of GLP-1 receptor in G-protein coupling2007Ingår i: Molecular Biology Reports, ISSN 0301-4851, E-ISSN 1573-4978, Vol. 34, nr 1, s. 53-60Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have demonstrated the ability of peptides derived from the third intracellular loop of GLP-1 receptor to differently modulate activity of four different types of G-proteins overexpressed in sf9 cells. In this respect, the involvement of Cys341 in inhibition of Gs and Cys341 in activation of Gs and in inhibition of Gi1, Go, and G11, respectively, indicates their potential role in discrimination between different types of G-proteins. Moreover, these two amino acids from the third intracellular loop might represent an important novel targets for covalent modification by downstream regulators in signaling through GLP-1 receptor.

  • 65.
    Bayat, Narges
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Toxicity and biological impact of metal and metal oxide nanoparticles: Focus on the vascular toxicity of ultra-small titanium dioxide nanoparticles2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The application of nanoparticles (NPs) in different technologies has led to tremendous advancement in those fields.  Moreover, there is growing interest in application of ultra-small NPs (USNPs) at 1-3 nm due to their distinct molecule like features. Parallel to these promises, there is a growing concern regarding their safety. The main goal of this thesis was to investigate the toxicity and underlying mechanisms following exposure to different metal and metal oxide NPs as well as USNPs. Their effects were studied on Saccharomyces cerevisiae, on hepatocytes and endothelial cells and finally in vivo on zebrafish embryos (Danio rerio). By selecting the rutile form of titanium dioxide (TiO2-USNPs) without intrinsic or intracellular reactive oxygen species (ROS) production, we could study biological impacts solely due to size and direct interaction with the cells. We showed that TiO2-USNPs were not cytotoxic but induced DNA damage. They had anti-angiogenic effects both in vitro and in vivo. Also, at high concentrations they caused complete mortality in zebrafish embryos exposed in water, while at lower concentrations induced delay in hatching. When injected they caused malformations. They specifically induced the differential overexpression of transcripts involved in lipid and cholesterol metabolism in endothelial cells. In hepatocytes they induced the overexpression of proteins in the electron transport chain and decreased lipids in lipid rafts. At 30 nm, TiO2-NPs, were also not cytotoxic but were genotoxic. They had no effects in vivo or on angiogenesis. However, they induced differential expression of transcripts involved in endoplasmic reticulum (ER) stress and heat shock response as well as cholesterol metabolism. This suggests a more toxic response in the cells compared to TiO2-USNPs.  Single walled carbon nanotubes (SWCNTs) despite having the highest oxidative activity among the NPs studied, were not severely cyto- or genotoxic but induced expression of transcripts involved in early ER stress response. Copper oxide (CuO-NPs) was the most toxic NPs studied due to both ion release and ROS production, affecting lipid metabolism of the cells. Silver (Ag-NPs) were also cytotoxic and caused the disruption of cellular components and lipids. ZnO-NPs were not cytotoxic, did not affect cellular lipids but they increased the size of vacuoles in yeast cells. Finally by using superparamagnetic iron oxide NPs (SPIONs) with different coatings, and using a mathematical model, a nano impact index (INI) was developed as a tool to enable the comparison of nanotoxicology data.

  • 66.
    Bayat, Narges
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cristobal, Susana
    The effects of ultra-small TiO2 nanoparticle and single walled carbon nanotubes on endothelial cells: next generation sequencing and transcriptome sequencing (RNA-seq) analysisManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The cardiovascular system is a key route of exposure to nanoparticles (NPs). The exposure could costendothelial cell dysfunction and impairment in blood circulation that could lead to cardiovascular diseasessuch as atherosclerosis. Currently, ultra-small nanoparticles (USNPs) at 1-3 nm, are receiving growingattention due to their unique properties. Emerging application for rutile TiO2-USNPs in medicine areexploring due to their insoluble nature, lack of oxidative activity and strong luminescence not observed inlarger NPs. On organic nanoparticle side, single walled carbon nanotubes (SWCNTs) are candidatemolecules for drug delivery from the chemical perspective. However their potential applications arehindered by their high oxidative activity and potential toxicity. Here we used transcriptome sequencing(RNA-seq) to evaluate the effects of exposure to sub-lethal concentration of TiO2-USNPs, TiO2-NPs andSWCNTs on human dermal microvascular endothelial cells. Specific toxicological effects were inferredfrom the functions of genes whose transcripts either increased or decreased. Our results show that TiO2-USNPs mostly induced the up-regulation of transcripts involved in lipid and cholesterol metabolism.TiO2-NPs induced the highest number of differentially expressed transcripts involved in cellularsenescence, endoplasmic reticulum (ER) stress, and heat shock responses as well lipid metabolism.Finally, SWCNTs affected to those genes involved in early stress and inflammatory responses.

  • 67.
    Bayat, Narges
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lopes, Viviana
    Sanchez-Dominguez, Maria
    Lakshmanan, Ramnath
    Rajarao, Gunaratna
    Cristobal, Susana
    Assessment of the safety of functionalized iron oxide nanoparticles in vitro: introduction to integrated nanoimpact indexManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Functionalization of super paramagnetic iron oxide NPs (SPIONs) with different coatings renders them with unique physicochemicalproperties that allow them to be used in a broad range of applications such as drug targeting and water purification. However, it is required tofill the gap between the promises of any new functionalized SPIONs and the effects of these coatings on the NPs safety. Nanotoxicology isoffering diverse strategies to assess the effect of exposure to SPIONs in a case-by-case manner but an integrated nanoimpact scale has notbeen developed yet. We have implemented the classical integrated biological response (IBR) into an integrated nanoimpact index (INI) as anearly warning scale of nano-impact based on a combination of toxicological end points such as cell proliferation, oxidative stress, apoptosisand genotoxicity. Here, the effect of SPIONs functionalized with tri-sodium citrate (TSC), polyethylenimine (PEI), aminopropyltriethoxysilane(APTES) and Chitosan (chitosan) were assessed on human keratinocytes and endothelial cells. Our results show thatendothelial cells were more sensitive to exposure than keratinocytes and the initial cell culture density modulated the toxicity. PEI-SPIONshad the strongest effects in both cell types while TSC-SPIONS were the most biocompatible. This study emphasizes not only the importanceof surface coatings but also the cell type and the initial cell density on the selection of toxicity assays. The INI developed here could offer aninitial rationale to choose either modifying SPIONs properties to reduce its nanoimpact or performing a complete risk assessment to definethe risk boundaries.

  • 68.
    Becedas, Luisa
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Xenobiotic-conjugating enzymes in rat ovary: hormonal regulation of UDP-glucuronosyltransferase and glutathione transferase and release of mutagenic metabolites by granulosa cells1997Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 69.
    Bendz, Maria
    et al.
    Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Skwark, Marcin
    Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, Daniel
    Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Granholm, Viktor
    Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cristobal, Susana
    Kall, Lukas
    Elofsson, Arne
    Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane protein shaving with thermolysin can be used to evaluate topology predictors2013Ingår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, nr 9, s. 1467-1480Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Topology analysis of membrane proteins can be obtained by enzymatic shaving in combination with MS identification of peptides. Ideally, such analysis could provide quite detailed information about the membrane spanning regions. Here, we examine the ability of some shaving enzymes to provide large-scale analysis of membrane proteome topologies. To compare different shaving enzymes, we first analyzed the detected peptides from two over-expressed proteins. Second, we analyzed the peptides from non-over-expressed Escherichia coli membrane proteins with known structure to evaluate the shaving methods. Finally, the identified peptides were used to test the accuracy of a number of topology predictors. At the end we suggest that the usage of thermolysin, an enzyme working at the natural pH of the cell for membrane shaving, is superior because: (i) we detect a similar number of peptides and proteins using thermolysin and trypsin; (ii) thermolysin shaving can be run at a natural pH and (iii) the incubation time is quite short. (iv) Fewer detected peptides from thermolysin shaving originate from the transmembrane regions. Using thermolysin shaving we can also provide a clear separation between the best and the less accurate topology predictors, indicating that using data from shaving can provide valuable information when developing new topology predictors.

  • 70.
    Bennett, Matthew
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Crystal structure of the essential biotin-dependent carboxylase AccA3 from Mycobacterium tuberculosis2017Ingår i: FEBS Open Bio, E-ISSN 2211-5463, Vol. 7, nr 5, s. 620-626Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Biotin-dependent acetyl-CoA carboxylases catalyze the committed step in type II fatty acid biosynthesis, the main route for production of membrane phospholipids in bacteria, and are considered a key target for antibacterial drug discovery. Here we describe the first structure of AccA3, an essential component of the acetyl-CoA carboxylase system in Mycobacterium tuberculosis (MTb). The structure, sequence comparisons, and modeling of ligand-bound states reveal that the ATP cosubstrate-binding site shows distinct differences compared to other bacterial and eukaryotic biotin carboxylases, including all human homologs. This suggests the possibility to design MTb AccA3 subtype-specific inhibitors.

  • 71.
    Bentinger, Magnus
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Karolinska Institutet.
    Kania, Magdalena
    Danikiewicz, Witold
    Kaczorowska, Ewa
    Wojcik, Jacek
    Brismar, Kerstin
    Dallner, Gustav
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Karolinska Institutet.
    Chojnacki, Tadeusz
    Swiezewska, Ewa
    Tekle, Michael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Karolinska Institutet.
    Effects of various squalene epoxides on coenzyme Q and cholesterol synthesis2014Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1841, nr 7, s. 977-986Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    2,3-Oxidosqualene is an intermediate in cholesterol biosynthesis and 2,3:22,23-dioxidosqualene act as the substrate for an alternative pathway that produces 24(S),25-epoxycholesterol which effects cholesterol homeostasis. In light of our previous findings concerning the biological effects of certain epoxidated all-trans-polyisoprenes, the effects of squalene carrying epoxy moieties on the second and third isoprene residues were investigated here. In cultures of HepG2 cells both monoepoxides of squalene and one of their hydrolytic products inhibited cholesterol synthesis and stimulated the synthesis of coenzyme Q (CoQ). Upon prolonged treatment the cholesterol content of these cells and its labeling with [H-3]mevalonate were reduced, while the amount and labeling of CoQ increased. Injection of the squalene monoepoxides into mice once daily for 6 days elevated the level of CoQ in their blood, but did not change the cholesterol level. The same effects were observed upon treatment of apoE-deficient mice and diabetic GK-rats. This treatment increased the hepatic level of CoQ10 in mice, but the amount of CoQ9, which is the major form, was unaffected. The presence of the active compounds in the blood was supported by the finding that cholesterol synthesis in the white blood cells was inhibited. Since the ratio of CoQ9/CoQ10 varies depending on the experimental conditions, the cells were titrated with substrate and inhibitors, leading to the conclusion that the intracellular isopentenyl-PP pool is a regulator of this ratio. Our present findings indicate that oxidosqualenes may be useful for stimulating both the synthesis and level of CoQ both in vitro and in vivo.

  • 72.
    Bentinger, Magnus
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Tekle, Michael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dallner, Gustav
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Coenzyme Q - Biosynthesis and functions2010Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 396, nr 1, s. 74-79Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In addition to its role as a component of the mitochondrial respiratory chain and our only lipid-soluble antioxidant synthesized endogenously, in recent years coenzyme Q (CoQ) has been found to have an increasing number of other important functions required for normal metabolic processes. A number of genetic mutations that reduce CoQ biosynthesis are associated with serious functional disturbances that can be eliminated by dietary administration of this lipid, making CoQ deficiencies the only mitochondrial diseases which can be successfully treated at present. In connection with certain other diseases associated with excessive oxidative stress, the level of CoQ is elevated as a protective response. Aging, certain experimental conditions and several human diseases reduce this level, resulting in serious metabolic disturbances. Since dietary uptake of this lipid is limited, up-regulation of its biosynthetic pathway is of considerable clinical interest. One approach for this purpose is administration of epoxidated all-trans polyisoprenoids, which enhance both CoQ biosynthesis and levels in experimental systems.

  • 73.
    Bentinger, Magnus
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Karolinska Institutet, Sweden.
    Tekle, Michael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Karolinska Institutet, Sweden.
    Dallner, Gustav
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Karolinska Institutet, Sweden.
    Brismar, Kerstin
    Gustafsson, Jan-Åke
    Steffensen, Knut R.
    Sergiu-Bogdan, Catrina
    Influence of liver-X-receptor on tissue cholesterol, coenzyme Q and dolichol content2012Ingår i: Molecular membrane biology, ISSN 0968-7688, E-ISSN 1464-5203, Vol. 29, nr 7, s. 299-308Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The organ content of the mevalonate pathway lipids was investigated in liver-X-receptor (LXR) alpha, beta and double knockout mice. An extensive or moderate increase of total cholesterol in the double KO mice was found in all organs elicited by the increase of the esterified form. In LXR alpha and double KO mice, coenzyme Q (CoQ) was decreased in liver and increased in spleen, thymus and lung, while dolichol was increased in all organs investigated. This effect was confirmed using LXR-agonist GW 3965. Analysis of CoQ distribution in organelles showed that the modifications are present in all cellular compartments and that the increase of the lipid in mitochondria was the result of a net increase of CoQ without changing the number of mitochondria. It appears that LXR influences not only cellular cholesterol homeostasis but also the metabolism of CoQ and dolichol, in an indirect manner.

  • 74. Berbalk, Christoph
    et al.
    Schwaiger, Christine S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lackner, Peter
    Accuracy analysis of multiple structure alignments2009Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 18, nr 10, s. 2027-2035Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Protein structure alignment methods are essential for many different challenges in protein science, such as the determination of relations between proteins in the fold space or the analysis and prediction of their biological function. A number of different pairwise and multiple structure alignment (MStA) programs have been developed and provided to the community. Prior knowledge of the expected alignment accuracy is desirable for the user of such tools. To retrieve an estimate of the performance of current structure alignment methods, we compiled a test suite taken from literature and the SISYPHUS database consisting of proteins that are difficult to align. Subsequently, different MStA programs were evaluated regarding alignment correctness and general limitations. The analysis shows that there are large differences in the success between the methods in terms of applicability and correctness. The latter ranges from 44 to 75% correct core positions. Taking only the best method result per test case this number increases to 84%. We conclude that the methods available are applicable to difficult cases, but also that there is still room for improvements in both, practicability and alignment correctness. An approach that combines the currently available methods supported by a proper score would be useful. Until then, a user should not rely on just a single program.

  • 75.
    Berg, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Proton transfer across and along biological membranes2020Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Proton-transfer reactions belong to the most prevalent reactions in the biosphere and make life on Earth possible, as they are central to energy conversion. In most known organisms, protons are translocated from one side of a membrane to the other, which generates an electrochemical gradient that drives ATP synthesis. Both the membranes and the proteins that are involved in these processes are vital components of energy-conversion machineries. This thesis presents and discusses proton transfer at surfaces of membranes and proteins, as well as proton translocation across membranes via enzymes.

    In the first work, we developed a single-enzyme approach to study proton translocation by the proton pump cytochrome bo3 (cyt. bo3). The generated proton gradients were stable as long as substrate (electrons, oxygen) was available. Individual cyt. bo3 could generate proton gradients of ∼2 pH units, which correspond to the measured electrochemical gradient in Escherichia coli cells.

    When acidic and basic amino acids are in close proximity to each other on a protein surface, their individual Coulomb cages can merge to form a proton antenna that enables fast proton transfer to specific groups. To investigate how the function of a proton pump is affected by structural changes in a proton antenna, close to a proton uptake pathway, we characterized the function and structure of genetic variants of cytochrome c oxidase (CytcO). When a Glu, located about 10 Å from the first residue of the D-pathway, was replaced by a non-protonatable residue (Ala) the proton pumping efficiency decreased by more than half compared to the wild-type enzyme. The proton-uptake kinetics was also altered in this variant.

    Cardiolipin (CL) is found in membranes where ATP is generated. This phospholipid alters the membrane structure and binds a variety of proteins including all complexes that take part in oxidative phosphorylation. To investigate the role of CL in proton-transfer reactions on the surface of membranes we used fluorescence correlation spectroscopy to study inner mitochondrial membranes from Saccharomyces cerevisiae. The protonation rate at wild-type membranes was about 50% of that measured with membranes prepared from mitochondria lacking CL. The protonation rate on the surface of small unilamellar vesicles (SUVs) decreased by about a factor of three when DOPC-SUVs were supplemented with 20% CL. Furthermore, phosphate buffer titrations with SUVs showed that CL can act as a local proton buffer in a membrane.

    The respiratory supercomplex factor 1 (Rcf1) has been suggested to facilitate direct electron transfer from the bc1 complex to CytcO by bridging the enzymes and binding cytochrome c (cyt. c) to a flexible domain of Rcf1. We investigated biding of cyt. c to Rcf1 reconstituted into different membrane environments. The apparent KD of the binding between cyt. c and DOPC-liposomes was almost five times lower when Rcf1 was present in the vesicles. Moreover, the apparent KD between cyt. c and liposome reconstituted CytcO was about nine times lower for CytcO isolated from a wild-type strain compared to a Rcf1-lacking strain.

  • 76.
    Berg, Johan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Liu, Jian
    Svahn, Emelie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ferguson-Miller, Shelagh
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structural changes at the surface of cytochrome c oxidase alter the proton-pumping stoichiometry2020Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1861, nr 2, artikel-id 148116Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Data from earlier studies showed that minor structural changes at the surface of cytochrome c oxidase, in one of the proton-input pathways (the D pathway), result in dramatically decreased activity and a lower proton-pumping stoichiometry. To further investigate how changes around the D pathway orifice influence functionality of the enzyme, here we modified the nearby C-terminal loop of subunit I of the Rhodobacter sphaeroides cytochrome c oxidase. Removal of 16 residues from this flexible surface loop resulted in a decrease in the proton-pumping stoichiometry to <50% of that of the wild-type enzyme. Replacement of the protonatable residue Glu552, part of the same loop, by an Ala, resulted in a similar decrease in the proton-pumping stoichiometry without loss of the O2-reduction activity or changes in the proton-uptake kinetics. The data show that minor structural changes at the orifice of the D pathway, at a distance of ~40 Å from the proton gate of cytochrome c oxidase, may alter the proton-pumping stoichiometry of the enzyme.

  • 77.
    Berg, Johan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sjöholm, Johannes
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bergstrand, Jan
    Widengren, Jerker
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The role of cardiolipin in lateral proton transfer along inner mitochondrial membranesManuskript (preprint) (Övrigt vetenskapligt)
  • 78. Berg, Stefan
    et al.
    Edman, Maria
    Li, Lu
    Wikström, Malin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes: Recognition of a large group of lipid glycosyltransferases in eubacteria and archaea2001Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, nr 25, s. 22056-22063Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Synthesis of the nonbilayer-prone α-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing properties and the                     lipid surface charge density in the membrane ofAcholeplasma laidlawii. The gene for the responsible, membrane-bound glucosyltransferase (alMGS) (EC 2.4.1.157) was sequenced and functionally cloned in Escherichia coli, yielding MGlcDAG in the recombinants. Similar amino acid sequences were encoded in the genomes of several Gram-positive                     bacteria (especially pathogens), thermophiles, archaea, and a few eukaryotes. All of these contained the typical EX7E catalytic motif of the CAZy family 4 of α-glycosyltransferases. The synthesis of MGlcDAG by a close sequence analog from                      Streptococcus pneumoniae (spMGS) was verified by polymerase chain reaction cloning, corroborating a connection between sequence and functional similarity                     for these proteins. However, alMGS and  spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol                     and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and                     spMGS) with the two-domain structure of the E. coli MurG cell envelope glycosyltransferase and several amphipathic membrane-binding segments in various proteins. On the basis                     of this structure, the alMGS sequence charge distribution, and anionic phospholipid dependence, a model for the bilayer surface                     binding and activity is proposed for this regulatory enzyme.

  • 79.
    Bergander, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Formation of formylindolo[3,2-b]carbazoles from tryptamine2007Konferensbidrag (Övrig (populärvetenskap, debatt, mm))
  • 80.
    Berglund, Anna-Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Spånning, Erika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biverståhl, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Maddalo, Gianluca
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    Tellgren-Roth, Christian
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dual targeting to Mitochondria and Chloroplasts: Characterization of Thr-tRNA Synthetase Targeting Peptide2009Ingår i: Molecular Plant, ISSN 1674-2052, Vol. 6, nr 2, s. 1298-1309Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins of Arabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides (dTPs). The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr–tRNA synthetase (ThrRS–dTP) studying organellar import of N- and C-terminal deletion constructs of ThrRS–dTP coupled to GFP. These results show that the 23 amino acid long N-terminal portion of ThrRS–dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS–dTP(2–60) to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS–dTP(2–60) inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS–dTP(2–60) indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.

  • 81.
    Bergquist, Helen
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Development of benzoquinoquinoxaline derivatives as triplex-specific probes: Recognition of DNA structures at repeats sequences2011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Repeat sequences are associated with several human diseases, such as Friedreich’s ataxia, polycystic kidney disease and cancer. These sequences can form non-B-DNA structures, including triplex (H-DNA) DNA, and are associated with genomic instability and altered gene expression. The occurrence of triplex structures in vivo and identification of their links to biological processes have been challenging. The lack of effective probes has restrained the study of triplex structures in living cells. Here, the triplex binding small molecule benzoquinoquinoxaline (BQQ) and its derivatives were developed as tools to study triplex formation at genomic repeat sequences. The triplex binding efficiency towards both purine and pyrimidine triplex motifs was determined for BQQ, the DNA cleaving BQQ-1,10-(ortho)-phenanthroline (BQQ-OP) and the fluorescent BQQ-Bodipy compounds. BQQ was shown to have the most stabilising effect on both triplex motifs. Moreover, H-DNA structure formation at a pkd1 derived sequence was demonstrated for the first time by BQQ-OP at physiologically relevant conditions. H-DNA formation was also shown at (GAA)n repeats associated with Friedreich’s ataxia and the structure was further analysed on one nucleotide resolution, confirming that (GAA)n repeats form a pyrimidine H-DNA. However, a mixture of different isomers formed at longer (GAA)n repeats. To this end, the interaction between the peptide nucleic acids (PNA) and BQQ was investigated. PNA is a DNA mimic that binds sequence-specifically to dsDNA and can form several PNA-DNA complexes. The results of PNA binding to frataxin (GAA)n expansion in plasmid were evaluated, and in the presence of GAA-PNA no triplex structure could be detected by BQQ-OP cleavage. When the structure formed in the presence of either GAA-PNA or CTT-PNA was further analysed, it was found that GAA-PNA formed a duplex invasion complex preventing H-DNA formation, whereas CTT-PNA formed a triplex invasion complex.

  • 82.
    Bergquist, Helen
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Jonsson, Niklas
    Nguyen, Chi-Hung
    Nielsen, Peter
    Good, Liam
    Zain, Rula
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    PNA sequence-specific binding of H-DNA forming Friedreich's ataxia (GAA)n repeatsManuskript (preprint) (Övrigt vetenskapligt)
  • 83.
    Bergquist, Helen
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Nikravesh, Abbas
    Fernández, Raquel Domingo
    Larsson, Veronica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Nguyen, Chi-Hung
    Good, Liam
    Zain, Rula
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Structure-Specific Recognition of Friedreich’s Ataxia (GAA)n Repeats by Benzoquinoquinoxaline Derivatives2009Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 10, nr 16, s. 2629-2637Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreich's ataxia. (GAA)nrepeats form non-B-DNA structures, including triple helix H-DNA and higher-order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non-B-DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)nbinding by triplex-stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex-DNA-cleaving BQQ-1,10-phenanthroline (BQQ-OP) compounds. We also examined the ability of these compounds to act as structural probes for H-DNA formation within higher-order structures at pathological frataxin sequences in plasmids. DNA-complex-formation analyses with a gel-mobility-shift assay and sequence-specific probing of H-DNA-forming (GAA)nsequences by single-strand oligonucleotides and triplex-directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)nrepeats under physiologically relevant conditions.

  • 84.
    Bergquist, Helen
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Sigurdsson, Susannah
    Percipalle, Piergiorgio
    Nguyen, Chi-Hung
    Sahlin, Margareta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Good, Liam
    Zain, Rula
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Pkd1 DNA triplex stabilization by benzoquinoquinoxaline derivativesArtikel i tidskrift (Refereegranskat)
  • 85.
    Bergqvist, Cecilia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Niss, Frida
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Beckman, Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Karolinska Institutet, Sweden.
    Maksel, Danuta
    Jafferali, Mohammed H.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kulyté, Agné
    Ström, Anna-Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Monitoring of chromatin organization in live cells by FRIC. Effects of the inner nuclear membrane protein Samp12019Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, nr 9, artikel-id e49Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In most cells, transcriptionally inactive heterochromatin is preferentially localized in the nuclear periphery and transcriptionally active euchromatin is localized in the nuclear interior. Different cell types display characteristic chromatin distribution patterns, which change dramatically during cell differentiation, proliferation, senescence and different pathological conditions. Chromatin organization has been extensively studied on a cell population level, but there is a need to understand dynamic reorganization of chromatin at the single cell level, especially in live cells. We have developed a novel image analysis tool that we term Fluorescence Ratiometric Imaging of Chromatin (FRIC) to quantitatively monitor dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation. We also found that peripheral heterochromatin depended on the levels of the inner nuclear membrane protein Samp1, suggesting an important role in promoting peripheral heterochromatin. Taken together, FRIC is a powerful and robust new tool to study dynamic chromatin redistribution in live cells.

  • 86. Berhe, Abraham
    Probing the function of the Pho84 high-affinity phosphate transporter of Saccharomyces cerevisiae2001Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 87.
    Bernsel, Andreas
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Viklund, Håkan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hennerdal, Aron
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    TOPCONS: consensus prediction of membrane protein topology2009Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, nr Suppl. 2, s. W465-W468Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    TOPCONS (http://topcons.net/) is a web server for consensus prediction of membrane protein topology. The underlying algorithm combines an arbitrary number of topology predictions into one consensus prediction and quantifies the reliability of the prediction based on the level of agreement between the underlying methods, both on the protein level and on the level of individual TM regions. Benchmarking the method shows that overall performance levels match the best available topology prediction methods, and for sequences with high reliability scores, performance is increased by approximately 10 percentage points. The web interface allows for constraining parts of the sequence to a known inside/outside location, and detailed results are displayed both graphically and in text format.

  • 88.
    Berntsson, Ronnie P. -A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Odegrip, Richard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Sehlén, Wilhelmina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Skaar, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Svensson, Linda M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Massad, Tariq
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Haggard-Ljungquist, Elisabeth
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Stenmark, Pål
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P22014Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, nr 4, s. 2725-2735Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 angstrom resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

  • 89. Bertaccini, Edward J.
    et al.
    Trudell, James R.
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Normal Mode Gating Motions of a Ligand-Gated Ion Channel Persist in a Fully Hydrated Lipid Bilayer Model2010Ingår i: ACS chemical neuroscience, ISSN 1948-7193, Vol. 1, nr 8, s. 552-558Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have previously used molecular modeling and normal-mode analyses combined with experimental data to visualize a plausible model of a transmembrane ligand-gated ion channel. We also postulated how the gating motion of the channel may be affected by the presence of various ligands, especially anesthetics. As is typical for normal-mode analyses, those studies were performed ut vacuo to reduce the computational complexity of the problem. While such calculations constitute an efficient way to model the large scale structural flexibility of transmembrane proteins, they can be criticized for neglecting the effects of an explicit phospholipid bilayer or hydrated environment. Here, we show the successful calculation of normal-mode motions for our model of a glycine alpha-1 receptor, now suspended in a fully hydrated lipid bilayer. Despite the almost uniform atomic density, the introduction of water and lipid does not grossly distort the overall gating motion. Normal-mode analysis revealed that even a fully immersed glycine alpha-1 receptor continues to demonstrate an iris-like channel gating motion as a low-frequency, high-amplitude natural harmonic vibration consistent with channel gating. Furthermore, the introduction of periodic boundary conditions allows the examination of simultaneous harmonic vibrations of lipid in synchrony with the protein gating motions that are compatible with reasonable lipid bilayer perturbations. While these perturbations tend to influence the overall protein motion, this work provides continued support for the iris-like motion model that characterizes gating within the family of ligand-gated ion channels.

  • 90.
    Berthold, Malin
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Galanin: ligand - receptor interactions1997Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 91.
    Bhuiyan, Hasanuzzaman
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Chromosome synapsis and recombination in yeast meiosis2004Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Meiosis is a cell division process that produces haploid gametes from diploid cells. Several important meiotic events take place during prophase of meiosis I, most important being homologous chromosome pairing, meiotic recombination and formation of the synaptonemal complex (SC). These processes assure proper segregation of the homologous chromosomes into the haploid germ cells. Improper segregation of the homologos can cause chromosomal abnormality (aneuploidy), which causes various human disorders, notably mental retardation and pregnancy loss.

    This thesis focuses on the relationship between recombination and the formation of SCs, aggregates of SC-related materials (polycomplexes) and recombination enzymes during meiosis. We have investigated SC formation in the absence of recombination, nature of polycomplexes and recombination enzymes in relation to the SCs structures and recombination nodules (RNs) in yeast Saccharomyces cerevisiae.

    Studies on yeast mutants suggest that the formation of SCs can take place only in the presence of the initiation of meiotic recombination through the action of the Spo11 enzyme. We have investigated the spo11 mutant of yeast that lacks initiation of meiotic recombination and observed that a fraction of this mutant cells (1.0%) can form complete SCs with wild-type appearance. We have further analyzed a spo11 mutant strain that accumalates at pachytene (spo11∆ndt80∆), and found that the frequency of cells with mature SC formation was 10%. The SC structures were detected in both immuno-fluorescence and electron microscopy (EM). Other phenotypic criteria such as spore viability and homologous chromosome pairing measured by FISH with chromosome specific probes, agrees with several previous reports of the spo11 mutant. Our results suggest that although synapsis is strongly promoted by Spo11 induced DSBs in yeast, it can take place in the absence initiation of meiotic recombination by Spo11 enzyme.

    In different organisms, the SCs are found to aggregate as stacks to form polycomplexes (PCs) that commonly occur before or after SC formation. It has generally been believed that the PCs are not attached to the chromosomes. We have investigated the ndt80 mutant of yeast that arrest at pachytene and found that although the SCs in spread chromosome preparations appear as wild type SCs, they appear as PCs in the intact nuclei in EM. In fluorescence in situ hybridization (FISH) with chromosome specific probes, we have shown that the homologous chromosomes in this mutant undergo high level of pairing and are attached to the SCs. In immuno-electron microscopy, two independent anti-DNA antibodies preferentially labeled the lateral element of the PCs. Our data suggests that the SCs in these polycomplexes can be involved in binding of chromosomes and are functional in pairing.

    The recombination enzymes, which are involved in the meiotic recombination process are believed to be components of recombination nodules. In some organisms, both early and late recombination enzymes have been shown to be located on RNs, but not in yeast before. We have studied immuno-localization and co-localization of Msh4, Msh5 and Sgs1 recombination enzymes on RNs in pachytene yeast nuclei with a newly developed EM method and found their co-localization on RNs along SCs. Msh4 and Msh5 are found to be located at the edges of RNs on the SCs. We have further analyzed the temporal appearance/disappearance and co-localization pattern of early and late meiotic recombination enzymes in relation to the SC development by immuno-fluorescence. We have been able to detect co-localization between the early meiosis specific enzyme Dmc1 and the late crossing-over related enzymes Msh5 as well as Sgs1. Our data suggest that Msh5 and Sgs1 are recruited to some sites of Dmc1, and thus erases the gap between early and late RNs.

  • 92.
    Bhushan, Shashi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    PresequenceProtease (PreP), a novel Peptidasome in Mitochondria and Chloroplasts: Localization, Function, Structure and Mechanism of Proteolysis2007Doktorsavhandling, monografi (Övrigt vetenskapligt)
    Abstract [en]

    The information for mitochondrial and chloroplastic protein import and targeting generally resides in the N-terminal part of the protein, called a targeting peptide. The targeting peptide is cleaved off by the organellar processing peptidases after import of a precursor protein. Free targeting peptides generated inside the organelle after import are rapidly degraded by proteolysis as their accumulation can have toxic effects on the organelle. The aim of this thesis has been to investigate the newly identified targeting peptide degrading protease, the PresequenceProtease (PreP). We have shown that the two isoforms of Arabidopsis PreP (AtPreP1 and AtPreP1) are dually targeted and localized to both mitochondria and chloroplasts. Dual targeting of the AtPreP1 is due to an ambiguous targeting peptide with a domain organization for mitochondrial and chloroplastic targeting. Both the AtPreP1 and AtPreP2 are expressed in Arabidopsis in an organ specific manner and they have distinct but overlapping substrate specificity. The crystal structure of the recombinant AtPreP1 E80Q was solved at 2.1 Å resolution. The structure represents the first substrate bound, closed conformation of a protease from the pitrilysin family. The PreP polypeptide folds in a unique peptidasome structure, surrounding a huge cavity of more than 10 000 Å3 in which the active site resides. A novel mechanism for proteolysis is proposed involving hinge-bending motions in response to substrate binding. PreP in human mitochondria has a novel function: degradation of amyloid β-peptide (Aβ). We show that under circumstances when Aβ is present in mitochondria of Alzheimer’s patients, PreP is the protease responsible for degradation of this toxic peptide.

  • 93.
    Bidla, Gawa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Genetic and molecular dissection of hemolymph coagulation and melanization in Drosophila melanogaster2007Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Injury to epithelial barriers puts metazoans at risk of loss of body fluid and contamination of their body by foreign particles. This risk is even exacerbated in insects, which have an open circulatory system and as a result, quickly need to seal wounds in order to keep a fairly constant internal milieu. Due to paucity of information on biochemical and molecular basis of insects’ clot, we studied how hemolymph of Drosophila melanogaster forms a clot, leading to a better understanding of responses after injury or infection in flies.

    By comparing hemolymph of Drosophila after bleeding with that described for an earlier model Galleria mellonella, we showed that a bona fide clot forms in Drosophila. The Drosophila clot is a fibrous network of crosslinked hemolymph proteins, which incorporates blood cells (plasmatocytes) extending shorter cellular processes of filopodia compared to cells outside the clot. Also, some plasmatocytes in the clot show features of apoptotic death while other blood cells (crystal cells) quickly rupture.

    The clot sequesters bacteria, as bacteria tethered to clot did not move. Clotting factors isolated include, Hemolectin (Hml) previously implicated in clotting, the immune induced protein Fondue and hemolymph proteins such as apolipophorin 2, fat body protein 1 and larval serum protein 1 γ. Hml mutants were more susceptible to infections when tested in a genetically sensitized background, suggesting that the clot may contribute to innate immunity. Clot also formed in hemolymph without phenoloxidase, an enzyme required for melanization and previously thought to be important for clot formation. However, we found that PO activity strengthens the clot to form a more solid plug.

    We found PO activity in clot to be induced in a transcription independent manner by inner membrane phospholipids: phosphatidylserine (PS) and phosphatidylinositol (PI) exposed on dead plasmatocytes and ruptured crystal cells. This is in contrast to induction of the enzyme during infection, which requires microbial components and transcriptional induction. However, both activation of PO in the clot and activation after infection appear to depend on proteases. Surprisingly, neither PS nor PI induced PO activity in the lepidopteran Galleria mellonella, in which the enzyme activity was instead induced by the microbial components peptidoglycan. This result may caution against generalizations of findings from using only one particular insect species. Finally, we found that the rupture of crystal cell during clot formation requires the Drosophila TNF homologue Eiger, JNK homologue Basket and small GTPases. This work therefore adds hemolymph clotting to the responses after injury or infection in flies and largely establishes Drosophila as a model to study coagulation of insect hemolymph. This will lead to a more comprehensive picture of Drosophila immunity with implications for other innate immune systems including our own.

  • 94.
    Björck, Markus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Regulation of proton-coupled electron transfer in cytochrome c oxidase: The role of membrane potential, proton pathways and ATP2019Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Cytochrome c oxidase (CytcO) is the final electron acceptor of the respiratory chain. In this chain a current of electrons, derived from degradation of nutrients, along with protons, are used to reduce oxygen to water. The reaction is exergonic and the excess energy is used to pump protons across the membrane. This proton-coupled electron transfer is regulated, for example, by the membrane potential, the composition of the membrane and the ATP/ADP concentrations. 

    Here, we have investigated the mechanism of this regulation. Specifically, we investigated ligand binding to CytcO in mitochondria, which provides mechanistic information about CytcO in its native environment. In addition to CytcO, a water soluble protein, flavohemoglobin (yHb) was found to bind CO and we found that it is localized in the intermembrane space (IMS). We also extracted CytcO from mitochondria without detergent using the styrene maleic acid (SMA) co-polymer. We could show that the SMA-extracted CytcO behaved similarly in its reaction with O2 and CO as CytcO in mitochondria.

    In mitochondria and bacterial membranes CytcO transports charges against a transmembrane electrochemical gradient. We induced a membrane potential across sub-mitochondrial particles (SMPs) by addition of ATP and measured single CytcO turnover. Our results indicate that proton transfer, but not electron transfer, across the membrane is affected by the membrane potential.

    In yeast CytcO subunit Cox13 has been shown to play a role in ATP/ADP binding to regulate activity. We have solved the structure of Cox13 using NMR and identified the residues that constitute the ATP-binding site, which is located at the C-terminus.

    Finally we showed that the main proton-transfer pathways in yeast CytcO function similarly to their bacterial counterparts and that the proposed H-pathway, absent in bacteria, is not responsible for proton translocation in mitochondrial CytcO from S. cerevisiae.

  • 95.
    Björck, Markus L.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vilhjálmsdóttir, Jóhanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hartley, Andrew M.
    Meunier, Brigitte
    Näsvik Öjemyr, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Maréchal, Amandine
    Brzezinski, Peter
    Proton-transfer pathways in the mitochondrial S. cerevisiae cytochrome c oxidaseManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    In cytochrome c oxidase (CytcO) reduction of O2 to water is linked to uptake of eight protons from the negative side of the membrane: four are substrate protons used to form water and four are pumped across the membrane. In bacterial oxidases, the substrate protons are taken up through the K and the D proton pathways, while the pumped protons are transferred through the D pathway. On the basis of studies with CytcO isolated from bovine heart mitochondria, it was suggested that in mitochondrial CytcOs the pumped protons are transferred though a third proton pathway, the H pathway, rather than throughthe D pathway. Here, we studied these reactions in S. cerevisiae CytcO, which serves as a model of the mammalian counterpart. We analyzed the effect of mutations in the D(Asn99Asp and Ile67Asn) and H pathways (Ser382Ala and Ser458Ala) and investigated the kinetics of electron and proton transfer during the reaction of the reduced CytcO withO2. No effects were observed with the H pathway variants while in the D pathway variants the functional effects were similar to those observed with the R. sphaeroides CytcO. The data indicate that the S. cerevisiae CytcO uses the D pathway for proton uptake and pumping.

  • 96. Björk, Behnosh
    et al.
    Pinho, Catarina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Alikhani, Nyosha
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bäckman, Hans
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Eneqvist, Therese
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Fratiglioni, Laura
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Graff, Caroline
    Genetic and biochemical studies of SNPs in the mitochondrial Aβ-degrading protease, hPrePManuskript (preprint) (Övrigt vetenskapligt)
  • 97.
    Björk Grimberg, Kristian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    The p97 ATPase and the Drosophila Proteasome: Protein Unfolding and Regulation2010Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    For all living systems, there is a requirement to recycle and regulate proteins. In eukaryotic organisms this is accomplished by the proteasome. The p97 ATPase is another highly conserved and essential complex present throughout the eukaryotic cell. In Paper I we utilized UFD fluorescent substrates to address the role of p97 and cofactors in soluble proteasome degradation. Results using RNAi and Drosophila p97 mutants propose p97 to function upstream of the proteasome on cytosolic proteasome targets as an important unfoldase together with its Ufd1/Npl4 cofactors. The results implicate p97 to be important for degradation of proteasome substrates lacking natural extended peptide regions. In Paper II we focused on identifying transcription factors essential for production of proteasomal subunits and associated proteins in Drosophila S2 cells. We utilized an RNA library targeting 993 known or candidate transcription factors and monitored RNAi depleted Drosophila S2 cells expressing the UFD reporter UbG76VGFP. We identified a range of potential candidates and focused on the bZIP transcription factor Cnc-C. RNAi and qrt-PCR experiments implicated Cnc-C to be involved in transcription of proteasomal subunits. In Paper III we applied our knowledge gained from Paper I about p97 dependent substrates and set up a high-throughput microscopy screening method to potentially find inhibitors specifically targeting the p97 proteasomal sub-pathway. Utilizing UFD substrates with and without C-terminal peptide tails we determined if compounds inhibited the core proteasomal machinery or the p97 pathway specifically. Through a primary and secondary round of screening we identified several new compounds inhibiting the ubiquitin-proteasome pathway though none from our initial screening had specificity for p97.

  • 98.
    Björk, Petra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Biogenesis of messenger and ribosomal RNPs in the eukaryotic cell2003Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In eukaryotic cells, gene expression involves multi-step processes in the nucleus and the cytoplasm. The different processes engage specific RNA-protein complexes, RNPs. Upon activation of most, if not all genes, a precursor RNA molecule is synthesized that has to be extensively processed and modified. In addition, the RNA has to associate with a distinct set of proteins. The composition of the RNP is often dynamic and changes over time. Several RNPs are exported to the cytoplasm, where they are involved in late steps of gene expression, including the synthesis of proteins.

    The aim of this thesis has been to increase our knowledge about specific steps in messenger RNP (mRNP) and ribosomal RNP (rRNP) biogenesis in the eukaryotic cell.

    We have characterized a novel protein, RBD-1, that is essential for ribosome biogenesis. RBD-1 contains six RNA-binding domains and is conserved in eukaryotes. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) is mainly located in the nucleolus, in an RNA polymerase I transcription-dependent manner. In cytoplasmic extracts, Ct-RBD-1 is preferentially associated with 40S ribosomal subunits. Ct-RBD-1 binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress prerRNA processing in vivo. RBD-1 is essential in Caenorhabditis elegans. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis.

    We have also studied the putative homologue to RBD-1 in Saccharomyces cerevisiae, Mrd1p. Mrd1p is essential for viability in yeast. Depletion of Mrd1p leads to a decrease in the synthesis of 18S rRNA and a decrease in the steady-state level of 40S ribosomal subunits. Mrd1p associates with prerRNA and U3 snoRNA and is required for the initial cleavages of the pre-rRNA. It is likely that Mrd1p is involved in the structural coordination of the pre-rRNA during early processing steps.

    We have identified and characterized the translation initiation factor eIF4H in the dipteran Chironomus tentans. We have studied its location and its relation to the transcription and translation processes. In the cytoplasm, Ct-eIF4H is associated with mRNA in polysomes. A minor fraction of CteIF4H is present in the nucleus, but it could not be detected in pre-mRNPs or mRNPs. The nuclear amount of Ct-eIF4H is independent of the level of transcription. We have addressed the question of where the translation machinery associates with mRNAs. Using immunoelectron microscopy, we can show that Ct-eIF4H associates with mRNPs in the cytoplasmic perinuclear region, immediately as the mRNP exits from the nuclear pore complex.

    Ct-RSF was isolated in a screen for RNA-binding proteins in Chironomus tentans. Ct-RSF has several properties in common with SR proteins, a family of conserved splicing factors. We have shown that Ct-RSF interacts with SR proteins, but in contrast to the splicing factors, Ct-RSF represses splicing in vitro. Our data suggest that Ct-RSF binds to exon sequences co-transcriptionally in vivo and that it represses the activation of splicing by SR proteins. It is conceivable that Ct-RSF is a protein that balances the action of SR proteins and avoids the formation of spliceosomes at aberrant splice sites in exons.

    Finally, we have initiated a study of Ct-Y14 and Ct-Mago. Pre-mRNA splicing deposits a multi-protein complex 20-24 nucleotides upstream of exon-exon junctions. This complex, the EJC, is believed to couple the splicing process to nuclear export, nonsense-mediated decay and cytoplasmic localization of mRNAs. The EJC contains a number of proteins, including Y14 and Mago. To contribute knowledge about the in vivo dynamics of Y14 and Mago, we decided to analyze the association of Y14 and Mago with the BR mRNAs in Chironomus tentans.

  • 99.
    Björk, Petra
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Wieslander, Lars
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Gene Expression in Polytene Nuclei2009Ingår i: Methods in Molecular Biology: The Nucleus / [ed] Ronald Hancock, Humana Press , 2009, 2, s. 29-53Kapitel i bok, del av antologi (Övrigt vetenskapligt)
    Abstract [en]

    Gene expression in eukaryotic cells is a multi-step process. Many of the steps are both co-ordinated and quality controlled. For example, transcription is closely coupled to pre-messenger RNA (mRNA)-protein assembly, pre-mRNA processing, surveillance of the correct synthesis of messenger ribonucleoprotein (mRNP), and export. The coordination appears to be exerted through dynamic interactions between components of the transcription, processing, surveillance, and export machineries. Our knowledge is so far incomplete about these molecular interactions and where in the nucleus they take place. It is therefore essential to analyze the intranuclear steps of gene expression in vivo. Polytene nuclei are exceptionally large and contain chromosomes and individual genes that can be structurally analyzed in situ during ongoing transcription. Furthermore, they contain gene-specific pre-mRNPs/mRNPs that can be visualised and analyzed as they are synthesised on the gene and then followed on their path to the cytoplasm. We describe methods for investigating the structure and composition of active chromatin and gene-specific pre-mRNPs/mRNPs in the context of analyses of gene expression processes in the nuclei of polytene cells.

  • 100.
    Björkander, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för immunologi.
    Bremme, K.
    Persson, Jan-Olov
    Stockholms universitet, Naturvetenskapliga fakulteten, Matematiska institutionen.
    van Vollenhoven, R. F.
    Sverremark-Ekström, Eva
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för immunologi.
    Holmlund, Ulrika
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för immunologi.
    Pregnancy-associated inflammatory markers are elevated in pregnant women with systemic lupus erythematosus2012Ingår i: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 59, nr 2, s. 392-399Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    During normal pregnancy a dampening in T cell-mediated immunity is compensated by an increased pro-inflammatory activity. Likewise, the autoimmune disease systemic lupus erythematosus (SLE) is associated with inflammatory activity and pregnancy complications occur frequently in women with SLE. The aim of this study was to elucidate how SLE influences the chemokine and cytokine balance during and after pregnancy. Blood samples were taken from pregnant women with or without SLE at second and third trimester and 8-12 weeks after pregnancy. Cytokines (interleukin (IL)-1 beta, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17A, TNF, IFN-gamma and IFN-alpha), chemokines (CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CCL2/MCP-1, CCL5/RANTES and CCL17/TARC), soluble IL-6 receptor (sIL-6R) and soluble glycoprotein 130 (gp130) were measured in serum using cytometric bead array (CBA) or enzyme-linked immunosorbent assay (ELISA). Women with SLE had increased serum concentrations of CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10 and IL-10 compared to controls both during and after pregnancy. Further, when dividing the patients based on disease activity, the women with active disease had the highest levels. Importantly, women with SLE seemed to respond to pregnancy in a similar way as controls, since the changes of cytokines and chemokines over the course of pregnancy were similar but with overall higher levels in the patient group. In conclusion, changes in pro- and anti-inflammatory serum components during pregnancy in women with SLE, occurring on top of already more pro-inflammatory levels, might increase their risk for pregnancy complications and flares. How their children are affected by this heightened inflammatory milieu during pregnancy needs further investigation.

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