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  • 301.
    Gubanova, Evgenia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Science for Life Laboratory; Division of Translational Medicine and Chemical Biology; Department of Medical Biochemistry and Biophysics; Karolinska Institut; Stockholm, Sweden.
    Issaeva, Natalia
    Djureinovic, Tatjana
    Helleday, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    SMG-1 regulates senescence and suppresses epithelial-mesenchymal transitionManuskript (preprint) (Övrigt vetenskapligt)
  • 302.
    Gubanova, Evgenia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Issaeva, Natalia
    Gokturk, Camilla
    Djureinovic, Tatjana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Helleday, Thomas
    SMG-1 suppresses CDK2 and tumor growth by regulating both the p53 and Cdc25A signaling pathways2013Ingår i: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 12, nr 24, s. 3770-3780Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The DNA damage response is coordinated by phosphatidylinositol 3-kinase-related kinases, ATM, ATR, and DNA-PK. SMG-1 is the least studied stress-responsive member of this family. Here, we show that SMG-1 regulates the G 1/S checkpoint through both a p53-dependent, and a p53-independent pathway. We identify Cdc25A as a new SMG-1 substrate, and show that cells depleted of SMG-1 exhibit prolonged Cdc25A stability, failing to inactivate CDK2 in response to radiation. Given an increased tumor growth following depletion of SMG-1, our data demonstrate a novel role for SMG-1 in regulating Cdc25A and suppressing oncogenic CDK2 driven proliferation, confirming SMG-1 as a tumor suppressor.

  • 303.
    Guell, Mireia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Luis, Josep M.
    Sola, Miquel
    Siegbahn, Per E. M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Theoretical study of the hydroxylation of phenolates by the Cu2O2(N,N '-dimethylethylenediamine)(2)(2+) complex2009Ingår i: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 14, nr 2, s. 229-242Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tyrosinase catalyzes the ortho hydroxylation of monophenols and the subsequent oxidation of the diphenolic products to the resulting quinones. In efforts to create biomimetic copper complexes that can oxidize C-H bonds, Stack and coworkers recently reported a synthetic mu-eta(2):eta(2)-peroxodicopper(II)(DBED)(2) complex ( DBED is N,N'-di-tert-butylethylenediamine), which rapidly hydroxylates phenolates. A reactive intermediate consistent with a bis-mu-oxo-dicopper(III)-phenolate complex, with the O-O bond fully cleaved, is observed experimentally. Overall, the evidence for sequential O-O bond cleavage and C-O bond formation in this synthetic complex suggests an alternative mechanism to the concerted or late-stage O-O bond scission generally accepted for the phenol hydroxylation reaction performed by tyrosinase. In this work, the reaction mechanism of this peroxodicopper(II) complex was studied with hybrid density functional methods by replacing DBED in the mu-eta(2):eta(2)-peroxodicopper(II)(DBED)(2) complex by N,N'-dimethylethylenediamine ligands to reduce the computational costs. The reaction mechanism obtained is compared with the existing proposals for the catalytic ortho hydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinone with the aim of gaining some understanding about the copper-promoted oxidation processes mediated by 2: 1 Cu(I)O-2-derived species.

  • 304.
    Gustafsson, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structural and functional studies of proteins of medical relevance: Protein-ligand complexes in cancer and novel structural folds in bacteria2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    X-ray crystallography is a tool for determining the structures of proteins and protein-ligand complexes. In this thesis the method has been employed to study several proteins of medical relevance.

    Cancer is a terrible disease, severely impacting those affected, as well as their family and friends. Current cancer treatments involve a combination of cytostatic drugs, surgery and radiation treatment. Unfortunately many cytostatic drugs also kill healthy cells, which gives rise to serious side-effects. The discovery of treatments which selectively inhibit proteins essential for cancer cell survival but which are non-essential in normal cells, could reduce such side-effects.

    MTH1 is a protein that degrades oxidised nucleotides, which when incorporated into DNA cause mutations and subsequent cell death. Cancer cells have higher levels of reactive oxygen species, which create oxidised nucleotides.  In Paper I it was discovered that cancer cells are dependent on MTH1 for their survival. Crystal structures of MTH1 in complex with small molecules guided their development into potent MTH1 inhibitors, capable of killing cancer cells. Cells with increased amounts of oxidised nucleotides, or with induced hypoxia, were more susceptible to MTH1 inhibition, as shown in Paper II. In Paper III several MTH1 orthologues from organisms often used in pre-clinical studies were tested for MTH1 inhibition. Leucine 116 of mouse MTH1 was determined to be important for the lower inhibition of the developed inhibitors towards this enzyme. A virtual fragment screening study using commercial chemicals resulted in several potent MTH1 inhibitors, as shown in Paper IV. The crystal structures with the fragments or optimised inhibitors did in most cases agree with the docking pose determined from the virtual screening. In addition to the known function of MTH1 in the degradation of oxidised nucleotides, Paper V showed that MTH1 also degrades methylated nucleotides.

    MTHFD2 is responsible for providing one-carbon units for nucleotide synthesis in cancer cells. As MTHFD2 is present in cancer cells but not in healthy cells, targeting the enzyme would make it possible to selectively kill cancer cells. Paper VI presents the first structure of MTHFD2, along with the first inhibitor of the protein. This information provides a starting point for the development of potent and selective MTHFD2 inhibitors.

    The botulinum neurotoxin from the bacterium Clostridium Botulinum is the causative agent of the deadly disease botulism. The action of the botulinum neurotoxin on nerve cells results in paralysis, and is life-threatening if the patient is not helped with breathing support. However, low doses of the neurotoxin are used as a successful treatment for several medical conditions, such as involuntary spasms. In Paper VII the structure of two proteins, P47 and OrfX2, encoded in the gene cluster of a botulinum neurotoxin, were determined. The structures resembled tubular lipid-binding proteins, previously only found in eukaryotes. The proteins were also found to be able to bind lipids. This work gives new insight into the structure and function of this group of proteins, which help the deadly botulinum neurotoxins.

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  • 305.
    Gustafsson, Robert
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berntsson, Ronnie P-A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Umeå University, Sweden.
    Martínez-Carranza, Markel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    El Tekle, Geniver
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Odegrip, Richard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Johnson, Eric A.
    Stenmark, Pål
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster2017Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, nr 22, s. 3781-3792Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 Å, respectively. We show that they belong to the TULIP protein superfamily, which are often involved in lipid binding. OrfX1 and OrfX2 were both found to bind phosphatidylinositol lipids.

  • 306. Gustafsson, Tomas N.
    et al.
    Sahlin, Margareta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Lu, Jun
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Holmgren, Arne
    Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase2012Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 47Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.

  • 307.
    Guterstam, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Characterization of cellular internalization pathways for CPP-mediated oligonucleotide delivery2011Ingår i: Cell-penetrating peptides: Methods and Protocols / [ed] Ülo Langel, New York: Humana Press, 2011, s. 219-230Kapitel i bok, del av antologi (Övrigt vetenskapligt)
    Abstract [en]

    The methods for evaluating internalization pathways of cellular CPP-mediated ON delivery utilizing a pre-mRNA splice correction assay and fluorescence-based quantification are described. Examples for characterization of CPP uptake routes, employing various endocytosis inhibitors, and special treatment conditions are demonstrated. The methods are developed to characterize cellular delivery of pre-mRNA splice switching peptide nucleic acids conjugated to CPPs by disulfide bond.

  • 308.
    Guterstam, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Madani, Fatemeh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hirose, Hisaaki
    Takeuchi, Toshihide
    Futaki, Shiroh
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate2009Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, nr 12, s. 2509-2517Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 309.
    Gyllberg, Hanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Prion-infection and Cellular Signaling: Influence of scrapie-infection on lipid raft-associated proteins2007Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Prion diseases are a group of fatal neurodegenerative diseases affecting almost all mammals including humans. The diseases are caused by formation of the misfolded isoform of the cellular prion protein (PrPC) to the disease causing PrPSc. The focus on this work has been to characterize molecular changes in persistently scrapie-infected murine neuronal cells possibly contributing to prion-induced neurodegeneration. PrPC is localized to lipid rafts in the plasma membrane and this is also the place where it is suggested that the conformational change into PrPSc occurs. This work shows an increased expression of active Src kinase in scrapie-infected cells resulting in an increased overall tyrosine phosphorylation of several proteins. Additionally, an increase in the specific tyrosine kinase activity of Fyn is shown. Interestingly, the membrane distribution of Fyn from non-raft to raft-domains followed that of PrPSc in scrapie-infected cells as analyzed by immunoblotting of flotation-fractions after ultracentrifugation of Triton X-100 extracted cell lysates. This indicates a persistent Fyn activation, probably due to clustering of intracellular Fyn kinases due to PrPSc accumulation in lipid rafts. In addition to an increased Src family kinase activity in scrapie-infected cells these cells also express an increased number of insulin receptor (IR)/insulin-like growth factor-1 receptor (IGF-1R) hybrid receptors, and these receptors display an altered protein glycosylation of the IR subunits. Additionally, ScN2a cells do not respond to LPS-stimulation with NO production, putatively due to the lack of CD14 mRNA. Together, these findings may have pathological implications leading to neuronal cell death in prion diseases via several mechanisms which are discussed in this thesis.

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  • 310.
    Gyllberg, Hanna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Löfgren, Kajsa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindegren, Heléne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bedecs, Katarina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Increased Src kinase level results in increased protein tyrosine phosphorylation in scrapie-infected neuronal cell lines.2006Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, nr 11, s. 2603-8Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have studied how prion infection may affect the Src kinase activity in three different neuronal cell lines, ScGT1 and ScN2a, where ScGT1 were generated in our laboratory. By immunoblotting, using clone 28 - a monoclonal antibody recognizing active Src, we have found a 32+/-6.3% and 75+/-7.7% elevation in Src activity in ScGT1 and ScN2a cells, respectively, compared to uninfected cells. Immunocomplex in vitro kinase assay confirmed the increased Src activity. The increased Src kinase activity in scrapie-infected cells was further shown to correlate to an increased level of Src protein. In addition, an important increase in the protein tyrosine phosphorylation signal was observed in ScGT1 and ScN2a cells, which was further shown to be Src-dependent, as treatment with PP2 - a Src family kinase specific inhibitor, reversed the protein tyrosine phosphorylation profile. Abnormal Src-kinase activation and subsequent protein tyrosine phosphorylation may be key elements in the neuropathology of the prion diseases.

  • 311.
    Götzke, Hansjörg
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Protein trafficking in the cell envelope of Escherichia coli: Identification and characterisation of a novel chaperone2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The cell envelope of Gram-negative bacteria, like Escherichia coli, is composed of a cytoplasmic membrane, a periplasmic space containing a peptidoglycan layer and an outer membrane. About 30 % of all proteins are localised in the cell envelope. These proteins have to be inserted into or translocated across the inner membrane by the SecYEG translocon. They are then chaperoned to their final destination by a network of chaperones. The broad aim of this work was to provide a better understanding of protein trafficking through the bacterial cell envelope. We have identified a novel membrane protein complex consisting of the periplasmic chaperone PpiD and the uncharacterised protein YfgM. Both are anchored in the inner membrane and have periplasmic domains. By co-immunoprecipitations and two-dimensional gel electrophoresis it could be demonstrated that YfgM and PpiD form a supercomplex with the SecYEG translocon. Furthermore, a chemical-genetic approach showed that YfgM is part of the periplasmic chaperone network that is essential for envelope protein biogenesis. Moreover, it could be shown that YfgM is required for the stability of the periplasmic chaperone HdeB. Finally, evidence that YfgM might also be involved in the lateral insertion of transmembrane domains was provided. In summary, this thesis details the identification and characterisation of a novel ancillary subunit of the SecYEG translocon that is involved in the periplasmic chaperone network in the cell envelope of Escherichia coli.

  • 312.
    Götzke, Hansjörg
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Muheim, Claudio
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Altelaar, A. F. Maarten
    Heck, Albert J. R.
    Maddalo, Gianluca
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Utrecht University, The Netherlands; Netherlands Proteomics Centre, The Netherlands.
    Daley, Daniel O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Identification of Putative Substrates for the Periplasmic Chaperone YfgM in Escherichia coli Using Quantitative Proteomics2015Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, nr 1, s. 216-226Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    How proteins are trafficked, folded, and assembled into functional units in the cell envelope of Gram-negative bacteria is of significant interest. A number of chaperones have been identified, however, the molecular roles of these chaperones are often enigmatic because it has been challenging to assign substrates. Recently we discovered a novel periplasmic chaperone, called YfgM, which associates with PpiD and the SecYEG translocon and operates in a network that contains Skp and SurA. The aim of the study presented here was to identify putative substrates of YfgM. We reasoned that substrates would be incorrectly folded or trafficked when YfgM was absent from the cell, and thus more prone to proteolysis (the loss-of-function rationale). We therefore used a comparative proteomic approach to identify cell envelope proteins that were lower in abundance in a strain lacking yfgM, and strains lacking yfgM together with either skp or surA. Sixteen putative substrates were identified. The list contained nine inner membrane proteins (CusS, EvgS, MalF, OsmC, TdcB, TdcC, WrbA, YfhB, and YtfH) and seven periplasmic proteins (HdeA, HdeB, AnsB, Ggt, MalE, YcgK, and YnjE), but it did not include any lipoproteins or outer membrane proteins. Significantly, AnsB (an asparaginase) and HdeB (a protein involved in the acid stress response), were lower in abundance in all three strains lacking yfgM. For both genes, we ruled out the possibility that they were transcriptionally down-regulated, so it is highly likely that the corresponding proteins are misfolded/mistargeted and turned-over in the absence of YfgM. For HdeB we validated this conclusion in a pulse-chase experiment. The identification of HdeB and other cell envelope proteins as potential substrates will be a valuable resource for follow-up experiments that aim to delineate molecular the function of YfgM.

  • 313.
    Götzke, Hansjörg
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Palombo, Isolde
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Muheim, Claudio
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Perrody, Elsa
    Genevaux, Pierre
    Kudva, Renuka
    Müller, Matthias
    Daley, Daniel O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    YfgM Is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli2014Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, nr 27, s. 19089-19097Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA.

  • 314. Hagey, Daniel W.
    et al.
    Zaouter, Cecile
    Combeau, Gaelle
    Andersson Lendahl, Monika
    Andersson, Olov
    Huss, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Muhr, Jonas
    Distinct transcription factor complexes act on a permissive chromatin landscape to establish regionalized gene expression in CNS stem cells2016Ingår i: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 26, nr 7, s. 908-917Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Spatially distinct gene expression profiles in neural stem cells (NSCs) are a prerequisite to the formation of neuronal diversity, but how these arise from the regulatory interactions between chromatin accessibility and transcription factor activity has remained unclear. Here, we demonstrate that, despite their distinct gene expression profiles, NSCs of the mouse cortex and spinal cord share the majority of their DNase I hypersensitive sites (DHSs). Regardless of this similarity, domain-specific gene expression is highly correlated with the relative accessibility of associated DHSs, as determined by sequence read density. Notably, the binding pattern of the general NSC transcription factor SOX2 is also largely cell type specific and coincides with an enrichment of LHX2 motifs in the cortex and HOXA9 motifs in the spinal cord. Interestingly, in a zebrafish reporter gene system, these motifs were critical determinants of patterned gene expression along the rostral-caudal axis. Our findings establish a predictive model for patterned NSC gene expression, whereby domain-specific expression of LHX2 and HOX proteins act on their target motifs within commonly accessible cis-regulatory regions to specify SOX2 binding. In turn, this binding correlates strongly with these DHSs relative accessibility-a robust predictor of neighboring gene expression.

  • 315.
    Haglund, Ellinor
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Folding of the Ribosomal protein S6: The role of sequence connectivity, overlapping foldons, and parallel pathways2009Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    To investigate how protein folding is affected by sequence connectivity five topological variants of the ribosomal protein S6 were constructed through circular permutation.  In these constructs, the chain connectivity (i.e. the order of secondary-structure elements) is changed without changing the native-state topology.  The effects of the permutations on the folding process were then characterised by φ-value analysis, which estimates the extent of contact formations in the transition-state ensemble.  The results show that the folding nuclei of the wild-type and permutant proteins comprises a common motif of one α-helix docking against two β-sheets, i.e. the minimal structure for folding.  However, this motif is recruited in different parts of the S6 structure depending on the permutation, either in the α1 or α2 half of the protein.  This minimal structure is not unique for S6 but can also be seen in other proteins.  As an effect of the dual nucleation possibilities, the transition-state changes describe a competition between two parallel pathways, which both include the central β-stand 1.  This strand constitutes thus a structural overlap between the two competing nuclei.  As similar overlap between competing nuclei is also seen in other proteins, I hypothesise that the coupling of several small nuclei into extended ‘super nuclei’ represents a general principle for propagating folding cooperativity across large structural distances.  Moreover, I demonstrate by NMR analysis that the existence of multiple folding nuclei renders the H/D-exchange kinetics independent of the folding pathway.

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  • 316.
    Haglund, Ellinor
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The HD-exchange motions of S6 are insensitive to reversal of the protein-folding pathwayManuskript (preprint) (Övrig (populärvetenskap, debatt, mm))
    Abstract [en]

    An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6 that comprises two folding nuclei (σ1 and σ2) defining two competing pathways in the folding energy landscape: s1→s2 and s2 →s1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate.

  • 317.
    Haglund, Ellinor
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öman, Tommy
    Department of chemistry, Umeå university.
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öhman, Anders
    Department of chemistry, Umeå university.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oliveberg, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The HD-exchange motions of ribosomal protein S6 are insensitive to reversal of the protein-folding pathway.2009Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 1091-6490, Vol. 106, nr 51, s. 21619-21624Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6, which comprises two folding nuclei (sigma1 and sigma2) defining two competing pathways in the folding energy landscape: sigma1 --> sigma2 and sigma2 --> sigma1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study, we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate.

  • 318.
    Hamada, Afaf M.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik. Assiut University.
    Jonsson, Lisbeth M. V.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik.
    Thiamine treatments alleviate aphid infestations in barley and pea2013Ingår i: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 94, s. 135-141Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Treatment of plants with thiamine (Vitamin B1) has before been shown to activate plant defence against microorganisms. Here, we have studied the effects of thiamine treatments of plants on aphid reproduction and behaviour. The work was mainly carried out with bird cherry-oat aphid (Rhopalosiphum padi L.) on barley (Hordeum vulgare L.). Aphid population growth and aphid acceptance on plants grown from seeds soaked in a 150 mu M thiamine solution were reduced to ca. 60% of that on control plants. R. padi life span and the total number of offspring were reduced on barley plants treated with thiamine. Healthy aphids and aphids infected with the R. padi virus were similarly affected. Spraying or addition of thiamine at 150 mu M to nutrient solutions likewise resulted in reduced aphid population growth to ca. 60%, as did plant exposure to thiamine odour at 4 mM. Thiamine treatments resulted in reduced aphid population growth also when tested with grain aphid (Sitobion avenae F.) on barley and pea aphid (Acyrthosiphon pisum H.) on pea (Pisum sativum L). There was no direct effect of thiamine on aphid reproduction or thiamine odour on aphid behaviour, as evaluated using artificial diets and by olfactometer tests, respectively. Two gene sequences regulated by salicylic acid showed higher transcript abundance and one gene sequence regulated by methyl jasmonate showed lower transcript abundance in thiamine-treated plants but not in control plants after aphid infestation. These results suggest that the aphid antibiosis and antixenosis effects may be related to priming of defence, but more studies are needed to explain the effects against aphids.

  • 319.
    Hamasur, Beston
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    F F₁-ATPase of plant mitochondria: purification and characterization of the structural and functional properties of the individual subunits1992Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Plant leaf cells constitute a unique system in which both mitochondria and chloroplasts are responsible for energy conversion. In both organelles, ATP synthase is responsible for the synthesis of ATP from ADP and Pi in a process coupled to an electron transfer chain.

    ATP synthase is a membrane-bound enzyme with a highly conserved structure in mitochondria and chloroplasts as well as in bacteria. The enzyme consists of two morphologically and functionally distinct domains, the catalytic Fi part and the H+-translocating F0 part. Although the chloroplast enzyme has been isolated and extensively characterized, difficulties associated with the purification of plant mitochondria contributed to the poor understanding of the plant mitochondrial ATP synthase. In the present study we have developed a large-scale purification procedure for spinach mitochondria almost free from thylakoid contaminants and, for the first time, purified and characterized the structural and catalytic properties of the plant mitochondrial ATP synthase.

    Isolation and purification of the spinach mitochondrial ATP synthase was achieved by a procedure involving 3-[(3-cholamidopropyl) dimethylammonio]-l-propanesulfonate (CHAPS) extraction of the enzyme from the inner mitochondrial membrane followed by density gradient centrifugation.

    The isolated complex consists of twelve polypeptides. Based on N-terminal amino acid sequence analysis and Western blotting using monospecific antibodies raised against proteins characterized in other sources, we identified five polypeptides of molecular masses 54 kDa (a,ß), 33 kDa (y), 21 kDa (8) and 9.5 kDa (e) as being the components of the catalytic Fi sector of the enzyme, and the remaining polypeptides of molecular masses of 28 kDa Qj), 23 kDa (OSCP), 18.5 kDa (a), 15 kDa (Fg), 10.5 kDa (lFi), 9.5 kDa (c) and 8.5 kDa (unknown) as the components of the F0 sector of the complex.

    Reconstitution experiments using Fi-depleted potato submitochondrial particles and the isolated potato subunits were used to study the role of the individual subunits of the complex. The Fj depleted particles were reconstitutively active. The 27 kDa and 23 kDa proteins which were found correspond to F0 subunit b. and OSCP, respectively, were also shown to be important for correct organization of the complex after reconstitution.

    A small, 9.5 kDa, loosely bound protein was identified as an endogenous ATPase inhibitor protein (lFi) and was found to play an important role in the regulation of ATPase activity of potato mitochondria. By the use of a simple procedure, this protein was for the first time purified and characterized from a plant source. Comparison of the potato inhibitor protein with those from other sources revealed the close structural and functional relationship between the plant and the yeast inhibitors.

    The FqFi complex in both membrane-bound and solublized forms catalyzes oligomycin-sensitive hydrolysis of the purine nucleotides in the order GTP > FTP > ATP. Hydrolysis of ATP, but not of other nucleotides was stimulated several fold in the presence of activators, such as anions and detergents. On the basis of these results, we classified the plant mitochondrial enzyme as a "similatent” ATPase.

    Translocation of the transmembrane proton gradient set up by respiration through the ATPase complex was measured in potato submitochondrial particles before and after depletion of Fi. Kinetic analysis of inhibition of the H+-translocation by oligomycin in both types of membranes showed an initial fast phase. Almost complete inhibition with 1 mol oligomycin per mol ATP synthase was observed in both types of particles, indicating that removal of Fi from the particles does not alter the kinetics of proton translocation through F0 and the interaction of F0 with oligomycin.

  • 320.
    Hansen, Ida
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Cannon, Barbara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Effects of differentiation on gene expression of certain brown adipocyte-secreted factorsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The possibility that brown adipose tissue can secrete factors to affect overall metabolism is a subject that attracts attention. Previous studies from our laboratory suggest that chemerin and collagen type III a1 (Col3a1) could be interesting factors secreted from brown adipose tissue.

    To evaluate chemerin and Col3a1 gene expression regulation, primary brown adipocyte cultures were stimulated with norepinephrine at different stages of differentiation. The present study shows that there was an effect of differentiation on gene expression of these factors but there was no effect of norepinephrine treatment.

    There has been a suggestion that different adenylate cyclases are responsible for regulating gene expression in brown adipocytes. However, neither differentiation nor norepinephrine treatment affected adenylate cyclase gene expression dramatically.

    The results suggest that norepinephrine is not the main regulator of gene expression of either of the two possible brown adipocyte-secreted factors, chemerin and Col3a1. Surprisingly, norepinephrine had no major effect on adenylate cyclase expression at any stage of differentiation.

  • 321.
    Hansen, Ida
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Jansson, Kim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Cannon, Barbara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Nedergaard, Jan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Contrasting effects of cold acclimation versus obesogenic diets onchemerin gene expression in brown and brite adipose tissuesManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Based on results from a signal sequence trap, we investigated chemerin gene expression in brown adipose tissue. Male NMRI mice were exposed to 30, 22 or 4 °C for 3 weeks, or were fed control (chow) diet, cafeteria diet or high-fat diet at thermoneutrality for the same time. In brown adipose tissue, cold acclimation strongly diminished chemerin gene expression, whereas obesogenic diets augmented expression. Qualitatively, changes in expression were paralleled in brite/beige adipose tissues (e.g. inguinal), whereas white adipose tissue (epididymal) did not react to these cues. Changes in tissue expression were not paralleled by alterations in plasma levels. The cellular regulation was not congruent with a sympathetic/adrenergic control of expression. The data are discussed in relation to suggested endocrine, paracrine and autocrine effects of this adipokine.

  • 322.
    Hansen, Ida
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Jansson, Kim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Cannon, Barbara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Nedergaard, Jan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Physiological effects on gene expression of some brown adipose tissue secreted factorsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Background: To investigate the role of brown adipose tissue as a secretory organ, a signal-sequence trap and microarray study were performed. Results indicated that adrenomedullin, collagen type 3 a1, lipocalin 2 and Niemann Pick type C 2 could be possible brown adipocyte-secreted proteins.

    Method: To investigate the physiological regulation of the gene expression of the adrenomedullin, collagen 3 a1, lipocalin 2 and Niemann Pick type C2 in brown adipose tissue, the following study was performed. Mice were exposed to cold or different high-caloric diets (high-fat diet and cafeteria diet) for three weeks and qPCR studies were performed of the genes of interest.

    Results & Conclusion: The study indicates that cold acclimation significantly suppressed gene expression of adrenomedullin, collagen type 3a and lipocalin 2 (P < 0.05). There was also an effect of the different diets but the results were not cohesive between the two high-caloric diets. However, none of the expression patterns between the cold-acclimated mice or the diet studies overlapped, suggesting that norepinephrine is not the key regulator of the gene expression of any of the investigated genes

  • 323.
    Hansen, Ida
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Ohgiya, Satoru
    Cannon, Barbara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Nedergaard, Jan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    A partial secretome of brown adipose tissueManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    To identify brown adipocyte secreted proteins a signal-sequence trap method was used. All genes identified were cloned and studied with microarray technique.

    The aim of this study was to evaluate how these genes were influenced under different physiological conditions, both in vivo and in vitro. Microarray studies were performed comparing primary brown adipocytes stimulated with norepinephrine to non-stimulated, and primary brown adipocytes compared to primary white adipocytes. In vivo studies were performed to evaluate physiological effects on gene expression in brown adipose tissue. Male NMRI mice were placed in cold or at thermoneutrality for 3 weeks and compared. Mice kept at room temperature were exposed to cafeteria diet for three weeks compared to regular diet.

    Results show that norepinephrine had effects on the expression of these potentially secreted genes. However, gene expression from physiological studies in vivo that could be expected to show similar expression patterns as norepinephrine-treated brown adipocytes did not do so. This indicates that other factors than norepinephrine can regulate gene expression of possible brown adipose tissue-secreted factors.

  • 324.
    Hansen, Ida R.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    The secretome of brown adipose tissue2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Brown adipose tissue has long been known for its heat-producing capacity, but less is known about its possible effects as a secretory organ. This thesis summarizes information about presently known factors secreted from brown adipose tissue and about their actions. We were able to add factors to the list by the use of a signal-sequence trap method. Results from the signal-sequence trap generated a list of suggested brown adipocyte secreted proteins; gene expression of these proteins was then further studied with microarray technique.

    One of the genes further analyzed was the adipokine chemerin. Gene expression of chemerin in brown adipose tissue was decreased in cold acclimation but increased with a high-caloric diet. This indicates that factors other than norepinephrine influence chemerin gene expression. The effects on chemerin gene expression were not be reflected in serum levels; therefore, chemerin secreted from brown adipose tissue is ascribed an autocrine/paracrine role.

    Signal-sequence trap and microarray studies suggested adrenomedullin, collagen type 3 a1, lipocalin 2 and Niemann Pick type C2 to be highly secreted from brown adipocytes. Gene expression of these factors was examined in vivo and in vitro. Our studies showed that both cold acclimation and high-caloric diet have an effect on gene expression of these factors. However, there was no effect on gene expression of chemerin and collagen type 3 a1 in norepinephrine-treated brown adipocyte cell cultures. This suggests that effects on gene expression of the examined possible brown adipocyte secreted proteins are not solely controlled by norepinephrine.

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  • 325.
    Hansson, Monika
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Abedi-Valugerdi, Manuchehr
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mercuric chloride induces a strong immune activation, but does not accelerate the development of dermal fibrosis in tight-skin 1 (Tsk 1) mice2004Ingår i: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 59, nr 5, s. 469-477Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In susceptible mice, mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of immunoglobulin (Ig) G1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. We have previously hypothesized that mercury confers more adverse immunological effects on those mouse strains, which are genetically prone to develop spontaneous autoimmune diseases than on normal strains. In this study, we tested our hypothesis in tight skin 1 (Tsk1/+) mice, a murine model for human scleroderma. As a support for our hypothesis, we observed that in Tsk1/+ mice, B cells were spontaneously hyperactive and that treatment with mercury induced a strong immune/autoimmune response in these mice, but not in their non-Tsk (+/+) littermates. This response was characterized by the formation of high numbers of splenic IgG1, IgG2b and IgG3 antibody-secreting cells, increased serum levels of IgE, production of IgG1 antibodies against single-stranded DNA (ssDNA), trinitrophenol (TNP) as well as thyroglobulin and the development of renal IgG1 deposits. Neither Tsk1/+ mice nor F1 hybrid crosses between this strain, and mercury susceptible B10.S (H-2s) were able to produce IgG1-ANolA in response to mercury. Moreover, mercury-induced immune activation in Tsk1/+ was not able to potentiate the progression of skin fibrosis in this strain. Thus, exposure to mercury accelerates the immune dysregulation, but not the development of skin fibrosis in Tsk1/+ mice.

  • 326. Hasmats, Johanna
    et al.
    Green, Henrik
    Solnestam, Beata Werne
    Zajac, Pawel
    Huss, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Orear, Cedric
    Validire, Pierre
    Bjursell, Magnus
    Lundeberg, Joakim
    Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples2012Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 425, nr 2, s. 379-383Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification. In this study we investigate region dropout using a 929 polymerase based WGA approach. DNA from 123 lung cancers specimens and corresponding normal tissue were used and evaluated by Sanger sequencing of the p53 exons 5-8. To enable comparative analysis of this scarce material, WGA samples were compared with unamplified material using a pooling strategy of the 123 samples. In addition, a more detailed analysis of exon 7 amplicons were performed followed by extensive cloning and Sanger sequencing. Interestingly, by comparing data from the pooled samples to the individually sequenced exon 7, we demonstrate that mutations are more easily recovered from WGA pools and this was also supported by simulations of different sequencing coverage. Overall this data indicate a limited random loss of genomic regions supporting the use of whole genome amplification for genomic analysis.

  • 327.
    Hayat, Sikander
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    BOCTOPUS: improved topology prediction of transmembrane β barrel proteins.2012Ingår i: Bioinformatics, ISSN 1460-2059, Vol. 28, nr 4, s. 516-522Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    MOTIVATION: Transmembrane β barrel proteins (TMBs) are found in the outer membrane of Gram-negative bacteria, chloroplast and mitochondria. They play a major role in the translocation machinery, pore formation, membrane anchoring and ion exchange. TMBs are also promising targets for antimicrobial drugs and vaccines. Given the difficulty in membrane protein structure determination, computational methods to identify TMBs and predict the topology of TMBs are important.

    RESULTS: Here, we present BOCTOPUS; an improved method for the topology prediction of TMBs by employing a combination of support vector machines (SVMs) and Hidden Markov Models (HMMs). The SVMs and HMMs account for local and global residue preferences, respectively. Based on a 10-fold cross-validation test, BOCTOPUS performs better than all existing methods, reaching a Q3 accuracy of 87%. Further, BOCTOPUS predicted the correct number of strands for 83% proteins in the dataset. BOCTOPUS might also help in reliable identification of TMBs by using it as an additional filter to methods specialized in this task.

    AVAILABILITY: BOCTOPUS is freely available as a web server at: http://boctopus.cbr.su.se/. The datasets used for training and evaluations are also available from this site.

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  • 328.
    Hayat, Sikander
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Ranking models of transmembrane beta-barrel proteins using Z-coordinate predictions2012Ingår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 28, nr 12, s. i90-I96Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Motivation: Transmembrane beta-barrels exist in the outer membrane of gram-negative bacteria as well as in chloroplast and mitochondria. They are often involved in transport processes and are promising antimicrobial drug targets. Structures of only a few beta-barrel protein families are known. Therefore, a method that could automatically generate such models would be valuable. The symmetrical arrangement of the barrels suggests that an approach based on idealized geometries may be successful. Results: Here, we present tobmodel; a method for generating 3D models of beta-barrel transmembrane proteins. First, alternative topologies are obtained from the BOCTOPUS topology predictor. Thereafter, several 3D models are constructed by using different angles of the beta-sheets. Finally, the best model is selected based on agreement with a novel predictor, ZPRED3, which predicts the distance from the center of the membrane for each residue, i.e. the Z-coordinate. The Z-coordinate prediction has an average error of 1.61 A. Tobmodel predicts the correct topology for 75% of the proteins in the dataset which is a slight improvement over BOCTOPUS alone. More importantly, however, tobmodel provides a C alpha template with an average RMSD of 7.24 A from the native structure.

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  • 329. Hayat, Sikander
    et al.
    Peters, Christoph
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Shu, Nanjiang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Tsirigos, Konstantinos D.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Inclusion of dyad-repeat pattern improves topology prediction of transmembrane beta-barrel proteins2016Ingår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 32, nr 10, s. 1571-1573Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Accurate topology prediction of transmembrane beta-barrels is still an open question. Here, we present BOCTOPUS2, an improved topology prediction method for transmembrane beta-barrels that can also identify the barrel domain, predict the topology and identify the orientation of residues in transmembrane beta-strands. The major novelty of BOCTOPUS2 is the use of the dyad-repeat pattern of lipid and pore facing residues observed in transmembrane beta-barrels. In a cross-validation test on a benchmark set of 42 proteins, BOCTOPUS2 predicts the correct topology in 69% of the proteins, an improvement of more than 10% over the best earlier method (BOCTOPUS) and in addition, it produces significantly fewer erroneous predictions on non-transmembrane beta-barrel proteins.

  • 330.
    Hedberg, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The birth and growth of the protein folding nucleus: Studies of protein folding focused on critical contacts, topology and ionic interactions2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Proteins are among the most complex molecules in the cell and they play a major role in life itself. The complexity is not restricted to just structure and function, but also embraces the protein folding reaction. Within the field of protein folding, the focus of this thesis is on the features of the folding transition state in terms of growing contacts, common nucleation motifs and the contribution of charged residues to stability and folding kinetics.

    During the resent decade, the importance of a certain residue in structure formation has been deduced from Φ-value analysis. As a complement to Φ-value analysis, I present how scatter in a Hammond plot is related to site-specific information of contact formation, Φ´(βTS), and this new formalism was experimentally tested on the protein L23. The results show that the contacts with highest Φ growth at the barrier top were distributed like a second layer outside the folding nucleus. This contact layer is the critical interactions needed to be formed to overcome the entropic barrier.

    Furthermore, the nature of the folding nucleus has been shown to be very similar among proteins with homologous structures and, in the split β-α-β family the proteins favour a two-strand-helix motif. Here I show that the two-strand-helix motif is also present in the ribosomal protein S6 from A. aeolicus even though the nucleation and core composition of this protein differ from other related structure-homologues.

    In contrast to nucleation and contact growth, which are events driven by the hydrophobic effect, my most recent work is focused on electrostatic effects. By pH titration and protein engineering the charge content of S6 from T. thermophilus was altered and the results show that the charged groups at the protein surface might not be crucial for protein stability but, indeed, have impact on folding kinetics. Furthermore, by site-specific removal of all acidic groups the entire pH dependence of protein stability was depleted.

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  • 331.
    Hedin, Linnea E
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Intra- and intermolecular interactions in proteins: Studies of marginally hydrophobic transmembrane alpha-helices and protein-protein interactions.2010Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Most of the processes in a living cell are carried out by proteins. Depending on the needs of the cell, different proteins will interact and form the molecular machines demanded for the moment. A subset of proteins called integral membrane proteins are responsible for the interchange of matter and information across the biological membrane, the lipid bilayer enveloping and defining the cell. Most of these proteins are co-translationally integrated into the membrane by the Sec translocation machinery.

    This thesis addresses two questions that have emerged during the last decade. The first concerns membrane proteins: a number of α-helices have been observed to span the membrane in the obtained three-dimensional structures even though these helices are predicted not to be hydrophobic enough to be recognized by the translocon for integration. We show for a number of these marginally hydrophobic protein segments that they indeed do not insert well outside of their native context, but that their local sequence context can improve the level of integration mediated by the translocon. We also find that many of these helices are overlapped by more hydrophobic segments. We propose, supported by experimental results, that the latter are initially integrated into the membrane, followed by post-translational structural rearrangements. Finally, we investigate whether the integration of the marginally hydrophobic TMHs of the lactose permease of Escherichia coli is facilitated by the formation of hairpin structures. However our combined efforts of computational simulations and experimental investigations find no evidence for this.

    The second question addressed in this thesis is that of the interpretation of the large datasets on which proteins that interact with each other in a cell. We have analyzed the results from several large-scale investigations concerning protein interactions in yeast and draw conclusions regarding the biases, strengths and weaknesses of these datasets and the methods used to obtain them.

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  • 332.
    Hedin, Linnea E.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Illergård, Kristoffer
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    An Introduction to Membrane Proteins2011Ingår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, nr 8, s. 3324-3331Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    alpha-Helical membrane proteins are important for many biological functions. Due to physicochemical constraints, the structures of membrane proteins differ from the structure of soluble proteins. Historically, membrane protein structures were assumed to be more or less two-dimensional, consisting of long, straight, membrane-spanning parallel helices packed against each other. However, during the past decade, a number of the new membrane protein structures cast doubt on this notion. Today, it is evident that the structures of many membrane proteins are equally complex as for many soluble proteins. Here, we review this development and discuss the consequences for our understanding of membrane protein biogenesis, folding, evolution, and bioinformatics.

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  • 333.
    Hedman, Rickard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dynamics of peptide chains during co-translational translocation, membrane integration & domain folding2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The biosynthesis of proteins occurs at the ribosomes, where amino acids are linked together into linear chains. Nascent protein chains may undergo several different processes during their synthesis. Some proteins begin to fold, while others interact with chaperones, targeting factors or processing enzymes. Nascent membrane proteins are targeted to the cell membrane for integration, which involves the translocation of periplasmic domains and the insertion of membrane-embedded parts.

    The aim of this thesis was to gain insights about the dynamics of nascent peptide chains undergoing folding, membrane translocation and integration. To this end, we explored the use of arrest peptides (APs) as force sensors. APs stall ribosomes when translated unless there is tension in the nascent peptide chain: the higher the tension, the more full-length protein can be detected. By using APs, we could show that a transmembrane helix is strongly ‘pulled’ twice on its way into the membrane and that strong electric forces act on negatively charged peptide segments translocating through the membrane. Furthermore, we discovered that APs could be used to detect protein folding and made the surprising discovery that a small protein domain folded well inside the ribosomal tunnel. Finally, we explored the arrest-stability of a large set of AP variants and found two extremely stable APs.

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  • 334. Hedskog, Louise
    et al.
    Brohede, Jesper
    Wiehager, Birgitta
    Pinho, Catarina Moreira
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Revathikumar, Priya
    Lilius, Lena
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Graff, Caroline
    Karlstrom, Helena
    Ankarcrona, Maria
    Biochemical studies of poly t variants in the alzheimer's disease associated tomm40 gene2012Ingår i: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 31, nr 3, s. 527-536Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The apolipoprotein E (APOE) gene remains the most strongly established risk factor for late onset Alzheimer's disease (LOAD). Recently the gene, TOMM40, which is in linkage disequilibrium with APOE, was identified to be associated with LOAD in genome-wide association studies. One of the identified polymorphisms in TOMM40 is rs10524523, which is located in intron 6 and composed of thymidine repeats varying between 14 to 36 base-pairs in length. Reported results are contradictory in regard to the very long poly-T variant that has been associated with both increased and decreased risk of LOAD. Our study aimed to elucidate the functional implication of rs10524523 in an in vitro model of human fibroblast cells obtained from cognitively healthy APOE epsilon 3/epsilon 4 carriers harboring very long or short poly-T variants coupled to their APOE epsilon 3 allele. We have studied (i) expression levels of TOM40 protein and mRNA, (ii) TOM40 mRNA splicing, and (iii) mitochondrial function and morphology; and we have found no significant differences in regards to very long or short poly-T variant.

  • 335.
    Hegazy, Usama M.
    et al.
    National Research Centre (NRC), Egypt.
    Musdal, Yaman
    Hacettepe University, Turkey.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hidden Allostery in Human Glutathione Transferase P1-1 Unveiled by Unnatural Amino Acid Substitutions and Inhibition Studies2013Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, nr 9, s. 1509-1514Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis-Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased kat and 300-fold increased K-M(GSH) values, displays an apparent Hill coefficient of 0.82 +/- 0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency k(cat)/K-M(GSH) to near the wild-type value but still yields Hill coefficients of 0.61 +/- 0.08 and 0.86 +/- 0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl-substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99 +/- 0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.

  • 336.
    Helmfors, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides: Uptake mechanism and the role of receptors2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Genes are the major regulators of biological processes in every living thing. Problems with gene regulation can cause serious problems for the organism; for example, most cancers have some kind of genetic component. Regulation of biological processes using oligonucleotides can potentially be a therapy for any ailment, not just cancer. The problem so far has been that the targets for oligonucleotide-based therapies all reside on the inside of cells, because the cellular plasma membrane is normally impermeable to large and charged molecules (such as oligonucleotides) a delivery method is needed. Cell-penetrating peptides are a class of carrier molecules that are able to induce the cellular membrane into taking them and their cargo molecules into the cells. Understanding how and why cell-penetrating peptides work is one of the first and most important steps towards improving them to the point where they become useful as carriers for oligonucleotide-based therapies. This thesis is comprised of four scientific papers that are steps toward finding an uptake mechanism for cell-penetrating peptides that have been non-covalently complexed with oligonucleotides. In Paper I, we show that the scavenger receptors are responsible for uptake of the cell-penetrating peptide PepFect14 in complex with a short single-stranded oligonucleotide. Paper II expands upon this first finding and shows that the same receptors are key players in the uptake of several other cell-penetrating peptides that have been complexed with either, long double-stranded plasmid DNA or short double-stranded RNA. Paper III improves the luciferase-based assay for short oligonucleotide delivery by increasing the throughput 4-fold and reducing the cost by 95 %. The fourth manuscript uses the assay developed in paper III to investigate the effects on cell-penetrating peptide-mediated delivery by each of the constituents of a 264-member library of ligands for G-protein coupled receptors. We identify three ligands that dose-dependently increase the luciferase expression compared to control cells. These three ligands are one positive-, one negative allosteric modulator of metabotropic glutamate receptor 5 and one antagonist of histamine receptor 3.

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  • 337.
    Helmfors, Henrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    GPCR-ligands influence the short oligonucleotide transfection efficacy of the cell-penetrating peptide; Pepfect14Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Cell-penetrating peptides can be used to deliver oligonucleotide-based cargoes into cells. We have previously shown that inhibition or knock-down of scavenger receptor type A results in a decreased oligonucleotide uptake. The remaining question is if the scavenger receptors are the only cell-surface receptors that can affect the uptake. By utilizing an optimized, higher throughput assay, for short oligonucleotide delivery using cell-penetrating peptides, and simultaneously adding a G-protein coupled receptor-ligand library. We show that two allosteric modulators (MPEP and VU 0357121) of metabotropic glutamate receptor type 5 and one histamine H3 receptor antagonist (Ciproxifan) have effects that can increase the transfection efficacy of PepFect in complex with a short single stranded oligonucleotide. Five different estrogen receptor ligands have negative effects on the transfection efficacy.

  • 338.
    Helmfors, Henrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Tartu University, Estonia.
    SCARA Involvement in the Uptake of Nanoparticles Formed by Cell-Penetrating Peptides2015Ingår i: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, Springer-Verlag New York, 2015, Vol. 1324, s. 163-174Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    The investigation of uptake mechanisms for cell-penetrating peptides (CPPs) is and has been an ongoing project for as long as the peptides have been known, a time period that now spans over two decades. The ultimate answer is yet to be revealed and the current understanding is that no "one" mechanism will ever be found. The reason for this is that the uptake mechanism seems to be dependent on a multitude of factors that include which CPP, what cells are used, whether or not there is cargo and what the cargo is. CPPs are capable of delivering a variety of bio-macromolecules that are by themselves unable to enter into cells. Our group has reported on many different peptides in recent years, many aimed at delivering various oligonucleotide-based cargoes. These peptides have utilized the inherent positive charge of the peptides and some rationally designed modifications to non-covalently complex oligonucleotides and bring them into cells. In this chapter, we present a brief overview of the current proposals for the uptake mechanisms of CPPs and describe methods for detecting and evaluating the role of scavenger receptor class A receptors in the uptake of non-covalent cell-penetrating peptide:oligonucleotide complexes.

  • 339.
    Hennerdal, Aron
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Application of membrane protein topology prediction2011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Membrane proteins often have essential functions in the cell and many are important drug targets, yet only a small fraction of available protein structures are of membrane proteins. Experimental techniques for elucidating membrane protein structures have proven laborious and expensive, opening the field for comparatively inexpensive computational modeling. Topology prediction addresses a sub-problem of structure prediction for α-helical membrane proteins by modeling which parts of the peptide chain are in, and which parts are on either side, of the membrane.

    This work describes an algorithm for combining the results of several topology prediction methods to increase prediction accuracy and to quantify prediction reliability, and a faster implementation of the algorithm applicable to large-scale genome data.

    Further, topology prediction is applied, together with other sequence-based methods, to detect duplications in membrane proteins in whole genomes. We find more duplications in the genomes of yeast and E. coli than in human, possibly due to the abundance of nonduplicated GPCRs in human. A gene duplication and subsequent fusion event constitute a likely origin for duplicated proteins, yet only for one superfamily, the AcrB Multidrug Efflux Pump, do we find the duplicated unit in its nonduplicated form. This apparent scarcity of nonduplicated forms is confirmed when extending the study to the whole human genome.

    Finally, a benchmark study of topology prediction on several comparably large datasets is described. We confirm previous results showing that methods utilizing homology information top the ranking of topology prediction methods. We also see that the separation of membrane proteins from non-membrane proteins has a partially different set of requirements than topology prediction of membrane proteins, and we suggest a pipeline using different methods for these two tasks.

  • 340.
    Hennerdal, Aron
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Falk, Jenny
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Internal duplications in alpha-helical membrane protein topologies are common but the nonduplicated forms are rare2010Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, nr 12, s. 2305-2318Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many alpha-helical membrane proteins contain internal symmetries, indicating that they might have evolved through a gene duplication and fusion event Here, we have characterized internal duplications among membrane proteins of known structure and in three complete genomes We found that the majority of large transmembrane (TM) proteins contain an internal duplication The duplications found showed a large variability both in the number of TM-segments included and in their orientation Surprisingly, an approximately equal number of antiparallel duplications and parallel duplications were found However, of all 11 superfamilies with an internal duplication, only for one, the AcrB Multidrug Efflux Pump, the duplicated unit could be found in its nonduplicated form An evolutionary analysis of the AcrB homologs indicates that several independent fusions have occurred, including the fusion of the SecD and SecF proteins into the 12-TM-protein SecDF in Brucella and Staphylococcus aureus In one additional case, the Vitamin B-12 transporter-like ABC transporters, the protein had undergone an additional fusion to form protein with 20 TM-helices in several bacterial genomes Finally, homologs to all human membrane proteins were used to detect the presence of duplicated and nonduplicated proteins This confirmed that only in rare cases can homologs with different duplication status be found, although internal symmetry is frequent among these proteins One possible explanation is that it is frequent that duplication and fusion events happen simultaneously and that there is almost always a strong selective advantage for the fused form

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  • 341.
    Henriksson, Linda
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Stenmark, Pål
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    pH dependence of the minimal progenitor complex of Botulinum neurotoxin XManuskript (preprint) (Övrigt vetenskapligt)
  • 342.
    Herman, Maria Dolores
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Moche, Martin
    Flodin, Susanne
    Welin, Martin
    Tresaugues, Lionel
    Johansson, Ida
    Nilsson, Martina
    Nordlund, Par
    Nyman, Tomas
    Structures of BIR domains from human NAIP and cIAP22009Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 65, s. 1091-1096Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1'-P4' side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3' position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2' and P4' pockets make similar interactions to those seen in other BIR domain-peptide complexes. The structures also reveal how a serine in the P1' position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins.

  • 343.
    Hernández-Neuta, Iván
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nucleic acid analysis tools: Novel technologies and biomedical applications2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Nucleic acids are fundamental molecules of living organisms functioning essentially as the molecular information carriers of life. From how an organism is built to how it responds to external conditions, all of it, can be found in the form of nucleic acid sequences inside every single cell of every life form on earth.

    Therefore, accessing these sequences provides key information regarding the molecular identity and functional state of any living organism, this is very useful for areas like biomedicine, where accessing and understanding these molecular signatures is the key to develop strategies to understand, treat and diagnose diseases.

    Decades of research and technological advancements have led to the development of a number of molecular tools and engineering technologies that allow accessing the information contained in the nucleic acids. This thesis provides a general overview of the tools and technologies available for nucleic acid analysis, and proposes an illustrative concept on how molecular tools and emergent technologies can be combined in a modular fashion to design methods for addressing different biomedical questions. The studies included in this thesis, are focused on the particular use of the molecular tools named: padlock and selector probes, rolling circle amplification, and fluorescence detection of single molecules in combination with microfluidics and portable microscopy. By using this combination, it became possible to design and demonstrate novel approaches for integrated nucleic acid analysis, inexpensive digital quantification, mobile-phone based diagnostics and the description of viral infections.

    These studies represent a step forward towards the adoption of the selected group of tools and technologies, for the design and building of methods that can be used as powerful alternatives to conventional tools used in molecular diagnostics and virology. 

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  • 344.
    Hernández-Neuta, Iván
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Pereiro, Iago
    Ahlford, Annika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Ferraro, Davide
    Zhang, Qiongdi
    Viovy, Jean-Louis
    Descroix, Stéphanie
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Microfluidic magnetic fluidized bed for DNA analysis in continuous flow modeManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA processing in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification (RCA). This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions and high throughput capabilities, with flow rates up to 5 L/min without compromising performance. The obtained efficiency values using the fluidized bed were superior to a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based micro arrayand tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostic systems.

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  • 345.
    Hessa, Tara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Integration of Transmembrane Helices into the Endoplasmic Reticulum2006Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Membrane proteins reside in cell and organelle membranes. They play significant roles in many processes vital to living cells. Receptors and ion channels are examples of membrane proteins that regulate the physiological state of the cell and are attractive targets for drug development.

    In eukaryotic cells most membrane proteins insert and fold cotranslationally into the endoplasmic reticular membrane. The insertion process is mediated by the Sec61 translocon which is a hetero-oligomeric protein-conducting channel that allows transmembrane segments to exit laterally into the lipid bilayer. How the translocon recognizes the molecular characteristics of transmembrane helices and integrate them into the lipid bilayer is the focus of this thesis.

    We have determined the sequence requirements for translocon-mediated integration of a transmembrane -helix into the ER membrane by challenging the Sec61 translocon with designed polypeptide segments in an in vitro expression system that permits quantitative assessment of membrane insertion efficiency. A biological hydrophobicity scale and a position-dependent free energy matrix have been developed, describing the contribution of each of the 20 amino acids in each position of a 19-residues long polypeptide segment to the overall free energy of a single transmembrane segment insertion. These studies suggest that the translocon provides direct contact between the nascent chain and the lipids in the membrane and that this protein-lipid interaction is the basis for the recognition of transmembrane helices in the translocon.

  • 346.
    Hessa, Tara
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bernsel, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sato, Yoko
    Lerch Bader, Mirjam
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    White, Stephen
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    A quantitative analysis of translocon-mediated insertion of transmembrane alpha-helicesManuskript (Övrigt vetenskapligt)
  • 347.
    Hessa, Tara
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kim, Hyun
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bihlmaier, Karl
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundin, Carolina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Boekel, Jorrit
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Andersson, Helena
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    White, Stephen
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Recognition of transmembrane helices by the endoplasmic reticulum translocon2005Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 433, nr 7024, s. 377-381Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein - lipid interactions are critical during translocon-mediated membrane insertion.

  • 348.
    Hessa, Tara
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Meindl-Beinker, Nadja M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bernsel, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kim, Hyun
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sato, Yoko
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lerch-Bader, Mirjam
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    White, Stephen H.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Molecular code for transmembrane-helix recognition by the Sec61 translocon2007Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 450, nr 7172, s. 1026-1030Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position- dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon- mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.

  • 349.
    Hessle, Viktoria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Characterization of RNA exosome in Insect Cells: Role in mRNA Surveillance2011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The exosome, an evolutionarily conserved protein complex with exoribonucleolytic activity, is one of the key players in mRNA quality control. Little is known about the functions of the exosome in metazoans. We have studied the role of the exosome in nuclear mRNA surveillance using Chironomus tentans and Drosophila melanogaster as model systems. Studies of the exosome subunits Rrp4 and Rrp6 revealed that both proteins are associated with transcribed genes and nascent pre-mRNPs in C. tentans. We have shown that several exosome subunits interact in vivo with the mRNA-binding protein Hrp59/hnRNP M, and that depleting Hrp59 in D. melanogaster S2 cells by RNAi leads to reduced levels of Rrp4 at the transcription sites. Our results on Rrp4 suggest a model for cotranscriptional quality control in which the exosome is constantly recruited to nascent mRNAs through interactions with specific hnRNP proteins. Moreover, we show that Rrp6 interacts with mRNPs in transit from the gene to the nuclear pore complex, where it is released during early stages of nucleo-cytoplasmic translocation. Furthermore, we show that Rrp6 is enriched in discrete nuclear bodies in the salivary glands of C. tentans and D. melanogaster. In C. tentans, the Rrp6-rich nuclear bodies colocalize with SUMO. We have also constructed D. melanogaster S2 cells expressing human b-globin genes, with either wild type of mutated splice sites, and we have studied the mechanisms by which the cells react to pre-mRNA processing defects. Our results indicate that two surveillance responses operate co-transcriptionally in S2 cells. One requires Rrp6 and retains defective mRNAs at the transcription site. The other one reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. These observations support the view that multiple mechanisms contribute to co-transcriptional surveillance in insects.

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  • 350.
    Hessle, Viktoria
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    von Euler, Anne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    de Valdivia, Ernesto Gonzalez
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore2012Ingår i: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 18, nr 8, s. 1466-1474Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Rrp6 is an exoribonuclease involved in the quality control of mRNA biogenesis. We have analyzed the association of Rrp6 with the Balbiani ring pre-mRNPs of Chironomus tentans to obtain insight into the role of Rrp6 in splicing surveillance. Rrp6 is recruited to transcribed genes and its distribution along the genes does not correlate with the positions of exons and introns. In the nucleoplasm, Rrp6 is bound to both unspliced and spliced transcripts. Rrp6 is released from the mRNPs in the vicinity of the nuclear pore before nucleo-cytoplasmic translocation. We show that Rrp6 is associated with newly synthesized transcripts during all the nuclear steps of gene expression and is associated with the transcripts independently of their splicing status. These observations suggest that the quality control of pre-mRNA splicing is not based on the selective recruitment of the exoribonuclease Rrp6 to unprocessed mRNAs.

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