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  • 201.
    Bagchi, Sonchita
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Tomenius, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Belova, Lyubov M.
    Ausmees, Nora
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces2008In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, no 4, p. 1037-1050Article in journal (Refereed)
    Abstract [en]

    Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)-like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF-like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF-like property to spontaneously assemble into filaments in vitro. Furthermore, the IF-like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF-like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks.

  • 202.
    Baiao, Guilherme Costa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Swedish Museum Nat Hist, Dept Zool, Stockholm, Sweden.
    Forshage, Mattias
    Swedish Museum Nat Hist, Dept Zool, Stockholm, Sweden.
    Revision of the West Palaearctic species of Rhoptromeris Forster, 1869 (Hymenoptera: Figitidae: Eucoilinae)2018In: Journal of Natural History, ISSN 0022-2933, E-ISSN 1464-5262, Vol. 52, no 17-18, p. 1201-1224Article in journal (Refereed)
    Abstract [en]

    The West Palearctic species of Rhoptromeris are revised. A total of 11 species are recognised as valid in this region, including four newly described species: Rhoptromeris dichromata sp. nov., Rhoptromeris koponeni sp. nov., Rhoptromeris leptocornis sp. nov. and Rhoptromeris macaronesiensis sp. nov. Eucoila luteicornis Ionescu, 1959 is synonymised with Rhoptromeris heptoma (Hartig, 1840) syn. nov. A checklist of the Holarctic Rhoptromeris is presented and an identification key to the West Palearctic species is provided.

    www.zoobank.org/urn:lsid:zoobank.org:pub:8164332C-93E2-4E3F-A408-F5FF5DFB366E

  • 203.
    Baiao, Guilherme Costa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Schneider, Daniela I.
    Med Univ Vienna, Ctr Anat & Cell Biol, Lab Genome Dynam, Deparment Cell & Dev Biol, Schwarzspanierstr 17, A-1090 Vienna, Austria;Yale Univ, Dept Epidemiol Microbial Dis, 60 Coll St, New Haven, CT 06510 USA.
    Miller, Wolfgang J.
    Med Univ Vienna, Ctr Anat & Cell Biol, Lab Genome Dynam, Deparment Cell & Dev Biol, Schwarzspanierstr 17, A-1090 Vienna, Austria.
    Klasson, Lisa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    The effect of Wolbachia on gene expression in Drosophila paulistorum and its implications for symbiont-induced host speciation2019In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 20, article id 465Article in journal (Refereed)
    Abstract [en]

    Background: The Neotropical fruit fly Drosophila paulistorum (Diptera: Drosophilidae) is a species complex in statu nascendi comprising six reproductively isolated semispecies, each harboring mutualistic Wolbachia strains. Although wild type flies of each semispecies are isolated from the others by both pre- and postmating incompatibilities, mating between semispecies and successful offspring development can be achieved once flies are treated with antibiotics to reduce Wolbachia titer. Here we use RNA-seq to study the impact of Wolbachia on D. paulistorum and investigate the hypothesis that the symbiont may play a role in host speciation. For that goal, we analyze samples of heads and abdomens of both sexes of the Amazonian, Centro American and Orinocan semispecies of D. paulistorum.

    Results: We identify between 175 and 1192 differentially expressed genes associated with a variety of biological processes that respond either globally or according to tissue, sex or condition in the three semispecies. Some of the functions associated with differentially expressed genes are known to be affected by Wolbachia in other species, such as metabolism and immunity, whereas others represent putative novel phenotypes involving muscular functions, pheromone signaling, and visual perception.

    Conclusions: Our results show that Wolbachia affect a large number of biological functions in D. paulistorum, particularly when present in high titer. We suggest that the significant metabolic impact of the infection on the host may cause several of the other putative and observed phenotypes. We also speculate that the observed differential expression of genes associated with chemical communication and reproduction may be associated with the emergence of pre- and postmating barriers between semispecies, which supports a role for Wolbachia in the speciation of D. paulistorum.

  • 204.
    Baier, Florian
    et al.
    Univ British Columbia, Michael Smith Lab, Vancouver, BC, Canada.
    Hong, Nansook
    Australian Natl Univ, Res Sch Chem, Canberra, ACT, Australia.
    Yang, Gloria
    Univ British Columbia, Michael Smith Lab, Vancouver, BC, Canada.
    Pabis, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Miton, Charlotte M.
    Univ British Columbia, Michael Smith Lab, Vancouver, BC, Canada.
    Barrozo, Alexandre
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Carr, Paul D.
    Australian Natl Univ, Res Sch Chem, Canberra, ACT, Australia.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Jackson, Colin J.
    Australian Natl Univ, Res Sch Chem, Canberra, ACT, Australia.
    Tokuriki, Nobuhiko
    Univ British Columbia, Michael Smith Lab, Vancouver, BC, Canada.
    Cryptic genetic variation shapes the adaptive evolutionary potential of enzymes2019In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e40789Article in journal (Refereed)
    Abstract [en]

    Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may impact the evolvability of proteins, but the underlying molecular basis remains unclear. Here, we performed comparative directed evolution of four orthologous metallo-beta-lactamases toward a new function and found that different starting genotypes evolved to distinct evolutionary outcomes. Despite a low initial fitness, one ortholog reached a significantly higher fitness plateau than its counterparts, via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show how cryptic molecular properties and conformational variation of active site residues in the initial genotypes cause epistasis, that could lead to distinct evolutionary outcomes. Our work highlights the importance of understanding the molecular details that connect genetic variation to protein function to improve the prediction of protein evolution.

  • 205. Bajt, Sasa
    et al.
    Chapman, Henry N
    Spiller, Eberhard A
    Alameda, Jennifer B
    Woods, Bruce W
    Frank, Matthias
    Bogan, Michael J
    Barty, Anton
    Boutet, Sebastien
    Marchesini, Stefano
    Hau-Riege, Stefan P
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Shapiro, David
    Camera for coherent diffractive imaging and holography with a soft-x-ray free-electron laser2008In: Applied Optics, ISSN 1559-128X, E-ISSN 2155-3165, Vol. 47, no 10, p. 1673-1683Article in journal (Refereed)
    Abstract [en]

    We describe a camera to record coherent scattering patterns with a soft-x-ray free-electron laser (FEL). The camera consists of a laterally graded multilayer mirror, which reflects the diffraction pattern onto a CCD detector. The mirror acts as a bandpass filter for both the wavelength and the angle, which isolates the desired scattering pattern from nonsample scattering or incoherent emission from the sample. The mirror also solves the particular problem of the extreme intensity of the FEL pulses, which are focused to greater than 10(14) W/cm2. The strong undiffracted pulse passes through a hole in the mirror and propagates onto a beam dump at a distance behind the instrument rather than interacting with a beam stop placed near the CCD. The camera concept is extendable for the full range of the fundamental wavelength of the free electron laser in Hamburg (FLASH) FEL (i.e., between 6 and 60 nm) and into the water window. We have fabricated and tested various multilayer mirrors for wavelengths of 32, 16, 13.5, and 4.5 nm. At the shorter wavelengths mirror roughness must be minimized to reduce scattering from the mirror. We have recorded over 30,000 diffraction patterns at the FLASH FEL with no observable mirror damage or degradation of performance.

  • 206.
    Baker, Brett J.
    et al.
    Univ Texas Austin, Inst Marine Sci, Dept Marine Sci, Port Aransas, TX 78373 USA..
    Saw, Jimmy H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lind, Anders E.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lazar, Cassandre Sara
    Univ Bremen, MARUM Ctr Marine Environm Sci, Bremen, Germany..
    Hinrichs, Kai-Uwe
    Univ Bremen, MARUM Ctr Marine Environm Sci, Bremen, Germany..
    Teske, Andreas P.
    Univ N Carolina, Dept Marine Sci, Chapel Hill, NC 27599 USA..
    Ettema, Thijs J. G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Genomic inference of the metabolism of cosmopolitan subsurface Archaea, Hadesarchaea2016In: Nature Microbiology, E-ISSN 2058-5276, Vol. 1, no 3, article id 16002Article in journal (Refereed)
    Abstract [en]

    The subsurface biosphere is largely unexplored and contains a broad diversity of uncultured microbes(1). Despite being one of the few prokaryotic lineages that is cosmopolitan in both the terrestrial and marine subsurface(2-4), the physiological and ecological roles of SAGMEG (South-African Gold Mine Miscellaneous Euryarchaeal Group) Archaea are unknown. Here, we report the metabolic capabilities of this enigmatic group as inferred from genomic reconstructions. Four high-quality (63-90% complete) genomes were obtained from White Oak River estuary and Yellowstone National Park hot spring sediment metagenomes. Phylogenomic analyses place SAGMEG Archaea as a deeply rooting sister clade of the Thermococci, leading us to propose the name Hadesarchaea for this new Archaeal class. With an estimated genome size of around 1.5 Mbp, the genomes of Hadesarchaea are distinctly streamlined, yet metabolically versatile. They share several physiological mechanisms with strict anaerobic Euryarchaeota. Several metabolic characteristics make them successful in the subsurface, including genes involved in CO and H-2 oxidation (or H-2 production), with potential coupling to nitrite reduction to ammonia (DNRA). This first glimpse into the metabolic capabilities of these cosmopolitan Archaea suggests they are mediating key geochemical processes and are specialized for survival in the subsurface biosphere.

  • 207. Balatsos, Nikolaos A. A.
    et al.
    Nilsson, Per
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mazza, Catherine
    Cusack, Stephen
    Virtanen, Anders
    Inhibition of mRNA Deadenylation by the Nuclear Cap Binding Complex (CBC)2006In: J. Biol. Chem., Vol. 281, p. 4517-4522Article in journal (Refereed)
  • 208.
    Balatsos, Nikolaos A A
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Cell Biology.
    Nilsson, Per
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Cell Biology.
    Mazza, Catherine
    Cusack, Stephen
    Virtanen, Anders
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Cell Biology.
    Inhibition of mRNA deadenylation by the nuclear cap binding complex (CBC).2006In: J Biol Chem, ISSN 0021-9258, Vol. 281, no 7, p. 4517-22Article in journal (Refereed)
  • 209.
    Ballante, Flavio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Protein-Ligand Docking in Drug Design: Performance Assessment and Binding-Pose Selection2018In: Rational Drug Design: Methods and Protocols / [ed] Thomas Mavromoustakos; Tahsin F. Kellici, New York, NY: Humana Press, 2018, p. 67-88Chapter in book (Refereed)
    Abstract [en]

    Main goal in drug discovery is the identification of drug-like compounds capable to modulate specific biological targets. Thus, the prediction of reliable binding poses of candidate ligands, through molecular docking simulations, represents a key step to be pursued in structure-based drug design (SBDD). Since the increasing number of resolved three-dimensional ligand-protein structures, together with the expansion of computational power and software development, the comprehensive and systematic use of experimental data can be proficiently employed to validate the docking performance. This allows to select and refine the protocol to adopt when predicting the binding pose of trial compounds in a target. Given the availability of multiple docking software, a comparative docking assessment in an early research stage represents a must-use step to minimize fails in molecular modeling. This chapter describes how to perform a docking assessment, using freely available tools, in a semiautomated fashion.

  • 210.
    Ballet, Caroline
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Correia, Mario S. P.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Conway, Louis P.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Locher, Theresa L.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Lehmann, Laura C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Garg, Neeraj
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vujasinovic, Miroslav
    Karolinska Univ Hosp, Dept Digest Dis, Stockholm, Sweden.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lohr, J. -Matthias
    Karolinska Inst, Dept Clin Sci Intervent & Technol CLINTEC, Stockholm, Sweden;Karolinska Univ Hosp, Dept Digest Dis, Stockholm, Sweden.
    Globisch, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery. Uppsala University, Science for Life Laboratory, SciLifeLab.
    New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites2018In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 9, no 29, p. 6233-6239Article in journal (Refereed)
    Abstract [en]

    Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.

  • 211. Balogh, Larissa M.
    et al.
    Le Trong, Isolde
    Kripps, A
    Tars, Kaspars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Stenkamp, E
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Atkins, William M.
    Structural Analysis of a Glutathione Transferase A1-1 Mutant Tailored for High Catalytic Efficiency with Toxic Alkenals2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 32, p. 7698-7704Article in journal (Refereed)
    Abstract [en]

    The specificity of human glutathione transferase (GST) A1-1 is drastically altered to favor alkenal substrates in the GIMFhelix mutant designed to mimic first-sphere interactions utilized by GSTA4-4. This redesign serves as a model for improving our understanding of the structural determinants that contribute to the distinct specificities of alpha class GSTs. Herein we report the first crystal structures of GIMFhelix, both in complex with GSH and in apo form at 1.98 and 2.38 angstrom resolution. In contrast to the preorganized hydrophobic binding pocket that accommodates alkenals in GSTA4-4, GSTA1-1 includes a dynamic alpha 9 helix that undergoes a ligand-dependent localization to complete the active site. Comparisons of the GIMFhelix structures with previously reported structures show a striking similarity with the GSTA4-4 active site obtained within an essentially GSTA1-1 scaffold and reveal the 0 helix assumes a similar localized structure regardless of active site occupancy in a manner resembling that of GSTA4-4. However, Are cannot fully account for all the structural elements important in GSTA4-4 within the mutant's active site. The contribution of Phe10 to the Tyr212-Phe10-Phe220 network prevents complete C-terminal Closure and demonstrates that the presence of Phe10 within the context of a GSTA4-4-like active site may ultimately hinder Phe220, a key C-terminal residue, from effectively contributing to the active site. In total, these results illustrate the remaining structural differences presumably reflected in the previously reported catalytic efficiencies of GIMFhelix and GSTA4-4 and emphasize the F10P mutation as being necessary to completely accomplish the transformation to a highly specific GST from the more promiscuous GSTA1-1 enzyme.

  • 212.
    Baltekin, Özden
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Astrego Diagnost AB, Uppsala, Sweden.
    KN Direct phenotypic antimicrobial susceptibility testing under 30 minutes: Dynamics of single cell response revealed in automated microscopy with microfluidics2018In: Journal of Veterinary Pharmacology and Therapeutics, ISSN 0140-7783, E-ISSN 1365-2885, Vol. 41, no S1, p. 17-17Article in journal (Other academic)
  • 213.
    Baltekin, Özden
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Boucharin, Alexis
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tano, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 34, p. 9170-9175Article in journal (Refereed)
    Abstract [en]

    The emergence and spread of antibiotic-resistant bacteria are aggravated by incorrect prescription and use of antibiotics. A core problem is that there is no sufficiently fast diagnostic test to guide correct antibiotic prescription at the point of care. Here, we investigate if it is possible to develop a point-of-care susceptibility test for urinary tract infection, a disease that 100 million women suffer from annually and that exhibits widespread antibiotic resistance. We capture bacterial cells directly from samples with low bacterial counts (10(4) cfu/mL) using a custom-designed microfluidic chip and monitor their individual growth rates using microscopy. By averaging the growth rate response to an antibiotic over many individual cells, we can push the detection time to the biological response time of the bacteria. We find that it is possible to detect changes in growth rate in response to each of nine antibiotics that are used to treat urinary tract infections in minutes. In a test of 49 clinical uropathogenic Escherichia coli (UPEC) isolates, all were correctly classified as susceptible or resistant to ciprofloxacin in less than 10 min. The total time for antibiotic susceptibility testing, from loading of sample to diagnostic readout, is less than 30 min, which allows the development of a point-of-care test that can guide correct treatment of urinary tract infection.

  • 214. Baltscheffsky, Herrick
    et al.
    Persson, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    On an Early Gene for Membrane-Integral Inorganic Pyrophosphatase in the Genome of an Apparently Pre-LUCA Extremophile, the Archaeon Candidatus Korarchaeum cryptofilum2014In: Journal of Molecular Evolution, ISSN 0022-2844, E-ISSN 1432-1432, Vol. 78, no 2, p. 140-147Article in journal (Refereed)
    Abstract [en]

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

  • 215.
    Baltzer, Nicholas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Komorowski, Jan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    ||-ROSETTAManuscript (preprint) (Other academic)
  • 216.
    Baltzer, Nicholas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Komorowski, Jan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sundström, Karin
    Nygård, Jan
    Nygård, Mari
    Dillner, Joakim
    Risk Stratification in Cervical Cancer Screening – Validation and Generalization of a Data-driven  Screening Recall ModelManuscript (preprint) (Other academic)
  • 217.
    Baltzer, Nicholas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Stockholm County, Sweden.
    Sundström, Karin
    Karolinska Inst, Dept Lab Med, Stockholm, Stockholm Count, Sweden..
    Nygård, Jan F.
    Canc Registry Norway, Dept Registry Informat, Oslo, Oslo County, Norway..
    Dillner, Joakim
    Karolinska Inst, Dept Lab Med, Stockholm, Stockholm Count, Sweden..
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Polish Acad Sci, Inst Comp Sci, Warsaw, Warsaw County, Poland..
    Risk stratification in cervical cancer screening by complete screening history: Applying bioinformatics to a general screening population2017In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 141, no 1, p. 200-209Article in journal (Refereed)
    Abstract [en]

    Women screened for cervical cancer in Sweden are currently treated under a one-size-fits-all programme, which has been successful in reducing the incidence of cervical cancer but does not use all of the participants' available medical information. This study aimed to use women's complete cervical screening histories to identify diagnostic patterns that may indicate an increased risk of developing cervical cancer. A nationwide case-control study was performed where cervical cancer screening data from 125,476 women with a maximum follow-up of 10 years were evaluated for patterns of SNOMED diagnoses. The cancer development risk was estimated for a number of different screening history patterns and expressed as Odds Ratios (OR), with a history of 4 benign cervical tests as reference, using logistic regression. The overall performance of the model was moderate (64% accuracy, 71% area under curve) with 61-62% of the study population showing no specific patterns associated with risk. However, predictions for high-risk groups as defined by screening history patterns were highly discriminatory with ORs ranging from 8 to 36. The model for computing risk performed consistently across different screening history lengths, and several patterns predicted cancer outcomes. The results show the presence of risk-increasing and risk-decreasing factors in the screening history. Thus it is feasible to identify subgroups based on their complete screening histories. Several high-risk subgroups identified might benefit from an increased screening density. Some low-risk subgroups identified could likely have a moderately reduced screening density without additional risk.

  • 218.
    Baltzer, Nicholas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sundström, Karin
    Karolinska Inst, Dept Lab Med, Stockholm, Sweden.
    Nygård, Jan
    Canc Registry Norway, Dept Registry Informat, Oslo, Norway.
    Nygård, Mari
    Canc Registry Norway, Dept Registry Informat, Oslo, Norway.
    Dillner, Joakim
    Karolinska Inst, Dept Lab Med, Stockholm, Sweden.
    Komorowski, Jan
    Uppsala Univ, Dept Cell & Mol Biol, Uppsala, Sweden;Polish Acad Sci, Warsaw, Poland.
    Stratifying Cervical Cancer Risk With Registry Data2018In: 2018 IEEE 14th International Conference on e-Science (e-Science 2018), IEEE, 2018, p. 288-289Conference paper (Refereed)
    Abstract [en]

    The cervical cancer screening programmes in Sweden and Norway have successfully reduced the frequency of cervical cancer incidence but have not implemented any form of evaluation for screening needs. This means that the screening frequency for individuals can he suboptimal, increasing either the cost of the programme or the risk of missing an early stage cancer development. We developed a framework for assessing an individual's risk of cervical cancer based on their available screening history and computing a primary risk factor called CRS from a data-driven separation model together with multiple derived attributes. The results show that this approach is highly practical, validates against multiple established trends, and can he effective in personalizing the screening needs for individuals.

  • 219.
    Balzarotti, Francisco
    et al.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Eilers, Yvan
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Gwosch, Klaus C.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Gynnå, Arvid H.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Westphal, Volker
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Stefani, Fernando D.
    Consejo Nacl Invest Cient & Tecn, Ctr Invest Bionanociencias CIBION, Buenos Aires, DF, Argentina.;Univ Buenos Aires, Fac Ciencias Exactas & Nat, Dept Fis, Buenos Aires, DF, Argentina..
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hell, Stefan W.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany.;Max Planck Inst Med Res, Dept Opt Nanoscopy, Heidelberg, Germany.;German Canc Res Ctr, Opt Nanoscopy Div, Heidelberg, Germany..
    Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes2017In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 355, no 6325, p. 606-612Article in journal (Refereed)
    Abstract [en]

    We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolutionmicroscopy, MINFLUXattained similar to 1-nanometer precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.

  • 220. Bandelt, HJ
    et al.
    Huber, KT
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, The Linnaeus Centre for Bioinformatics.
    Moulton, V
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, The Linnaeus Centre for Bioinformatics.
    Quasi-median graphs from sets of partitions2002In: Discrete Applied Mathematics, ISSN 0166-218X, Vol. 122, no 23-35, p. 23-35Article in journal (Other (popular scientific, debate etc.))
    Abstract [en]

    In studies of molecular evolution, one is typically confronted with the task of inferring a phylogenetic tree from a set X of sequences of length n over a finite alphabet Lambda. For studies that invoke parsimony, it has been found helpful to consider the quasi-median graph generated by X in the Hamming graph Lambda(n). Although a great deal is already known about quasi-median graphs (and their algebraic counterparts), little is known about the quasi-median generation in Lambda(n) starting from a set X of vertices. We describe the vertices of the quasi-median graph generated by X in terms of the coordinatewise partitions of X. In particular, we clarify when the generated quasi-median graph is the so-called relation graph associated with X. This immediately characterizes the instances where either a block graph or the total Hamming graph is generated.

  • 221.
    Banerjee, Debapriya
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Protein Folding Activity of the Ribosome (PFAR): A Target for Antiprion Compounds2014In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 6, no 10, p. 3907-3924Article, review/survey (Refereed)
    Abstract [en]

    Prion diseases are fatal neurodegenerative diseases affecting mammals. Prions are misfolded amyloid aggregates of the prion protein (PrP), which form when the alpha helical, soluble form of PrP converts to an aggregation-prone, beta sheet form. Thus, prions originate as protein folding problems. The discovery of yeast prion(s) and the development of a red-/white-colony based assay facilitated safe and high-throughput screening of antiprion compounds. With this assay three antiprion compounds; 6-aminophenanthridine (6AP), guanabenz acetate (GA), and imiquimod (IQ) have been identified. Biochemical and genetic studies reveal that these compounds target ribosomal RNA (rRNA) and inhibit specifically the protein folding activity of the ribosome (PFAR). The domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active site for PFAR. 6AP and GA inhibit PFAR by competition with the protein substrates for the common binding sites on the domain V rRNA. PFAR inhibition by these antiprion compounds opens up new possibilities for understanding prion formation, propagation and the role of the ribosome therein. In this review, we summarize and analyze the correlation between PFAR and prion processes using the antiprion compounds as tools.

  • 222.
    Banerjee, Debapriya
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Vovusha, Hakkim
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pang, Yanhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Oumata, Nassima
    Sanyal, Biplab
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Spectroscopic and DFT studies on 6-Aminophenanthridine and its derivatives provide insights in their activity towards ribosomal RNA2014In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 97, p. 194-199Article in journal (Refereed)
    Abstract [en]

    6-Aminophenanthridine (6AP), a plant alkaloid possessing antiprion activity, inhibits ribosomal RNA dependent protein folding activity of the ribosome (referred as PFAR). We have compared 6AP and its three derivatives 6AP8Cl, 6AP8CF3 and 6APi for their activity in inhibition of PFAR. Since PFAR inhibition requires 6AP and its derivatives to bind to the ribosomal RNA (rRNA), we have measured the binding affinity of these molecules to domain V of 23S rRNA using fluorescence spectroscopy. Our results show that similar to the antiprion activity, both the inhibition of PFAR and the affinity towards rRNA follow the order 6AP8CF3 > 6AP8Cl > 6AP, while 6APi is totally inactive. To have a molecular insight for the difference in activity despite similarities in structure, we have calculated the nucleus independent chemical shift using first principles density functional theory. The result suggests that the deviation of planarity in 6APi and steric hindrance from its bulky side chain are the probable reasons which prevent it from interacting with rRNA. Finally, we suggest a probable mode of action of 6AP, 6AP8CF3 and 6AP8Cl towards rRNA.

  • 223.
    Bang S, Koolen JH, Moulton V
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, The Linnaeus Centre for Bioinformatics.
    A bound for the number of columns l((c,a,b)) in the intersection array of a distance-regular graph2003In: European Journal of Combinatorics, ISSN 0195-6698, Vol. 24, no 7, p. 785-795Article in journal (Refereed)
  • 224.
    Baranowska, Izabella
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jäderlund, Karin Hultin
    Nennesmo, Inger
    Holmqvist, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Heidrich, Nadja
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Larsson, Nils-Göran
    Andersson, Göran
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hedhammar, Åke
    Wibom, Rolf
    Andersson, Leif
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sensory ataxic neuropathy in golden retriever dogs is caused by a deletion in the mitochondrial tRNATyr gene2009In: PLoS Genetics, ISSN 1553-7390, Vol. 5, no 5, p. e1000499-Article in journal (Refereed)
    Abstract [en]

    Sensory ataxic neuropathy (SAN) is a recently identified neurological disorder in golden retrievers. Pedigree analysis revealed that all affected dogs belong to one maternal lineage, and a statistical analysis showed that the disorder has a mitochondrial origin. A one base pair deletion in the mitochondrial tRNA(Tyr) gene was identified at position 5304 in affected dogs after re-sequencing the complete mitochondrial genome of seven individuals. The deletion was not found among dogs representing 18 different breeds or in six wolves, ruling out this as a common polymorphism. The mutation could be traced back to a common ancestor of all affected dogs that lived in the 1970s. We used a quantitative oligonucleotide ligation assay to establish the degree of heteroplasmy in blood and tissue samples from affected dogs and controls. Affected dogs and their first to fourth degree relatives had 0-11% wild-type (wt) sequence, while more distant relatives ranged between 5% and 60% wt sequence and all unrelated golden retrievers had 100% wt sequence. Northern blot analysis showed that tRNA(Tyr) had a 10-fold lower steady-state level in affected dogs compared with controls. Four out of five affected dogs showed decreases in mitochondrial ATP production rates and respiratory chain enzyme activities together with morphological alterations in muscle tissue, resembling the changes reported in human mitochondrial pathology. Altogether, these results provide conclusive evidence that the deletion in the mitochondrial tRNA(Tyr) gene is the causative mutation for SAN.

  • 225. Barbaro, Michela
    et al.
    Soardi, Fernanda C.
    Ostberg, Linus J.
    Persson, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    de Mello, Maricilda Palandi
    Wedell, Anna
    Lajic, Svetlana
    In vitro functional studies of rare CYP21A2 mutations and establishment of an activity gradient for nonclassic mutations improve phenotype predictions in congenital adrenal hyperplasia2015In: Clinical Endocrinology, ISSN 0300-0664, E-ISSN 1365-2265, Vol. 82, no 1, p. 37-44Article in journal (Refereed)
    Abstract [en]

    BackgroundA detailed genotype-phenotype evaluation is presented by studying the enzyme activities of five rare amino acid substitutions (Arg233Gly, Ala265Ser, Arg341Trp, Arg366Cys and Met473Ile) identified in the CYP21A2 gene in patients investigated for Congenital adrenal hyperplasia (CAH). ObjectiveTo investigate whether the mutations identified in the CYP21A2 gene are disease causing and to establish a gradient for the degree of enzyme impairment to improve prediction of patient phenotype. Design and patientsThe CYP21A2 genes of seven patients investigated for CAH were sequenced and five mutations were identified. The mutant proteins were expressed in vitro in COS-1 cells, and the enzyme activities towards the two natural substrates were determined to verify the disease-causing state of the mutations. The in vitro activities of these rare mutations were also compared with the activities of four mutations known to cause nonclassic CAH (Pro30Leu, Val281Leu, Pro453Ser and Pro482Ser) in addition to an in silico structural evaluation of the novel mutants. Main outcome measureTo verify the disease-causing state of novel mutations. ResultsFive CYP21A2 mutations were identified (Arg233Gly, Ala265Ser, Arg341Trp, Arg366Cys and Met473Ile). All mutant proteins exhibited enzyme activities above 5%, and four mutations were classified as nonclassic and one as a normal variant. By comparing the investigated protein changes with four common mutations causing nonclassic CAH, a gradient for the degree of enzyme impairment could be established. Studying rare mutations in CAH increases our knowledge regarding the molecular mechanisms that render a mutation pathogenic. It also improves phenotype predictions and genetic counselling for future generations.

  • 226.
    Barlow, Nicholas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052, Australia.
    Vanga, Sudarsana Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sävmarker, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Sandström, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Burns, Peta
    Biomedicine Discovery Institute, Department of Physiology, Monash University, Clayton, Victoria 3800, Australia.
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hallberg, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, BMC, SE-751 24 Uppsala, Sweden.
    Chai, Siew Yeen
    Biomedicine Discovery Institute, Department of Physiology, Monash University, Clayton, Victoria 3800, Australia.
    Thompson, Philip E
    Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052, Australia.
    Macrocyclic Peptidomimetics as Inhibitors of Insulin-Regulated Aminopeptidase (IRAP)Manuscript (preprint) (Other academic)
    Abstract [en]

    Macrocyclic analogues of the linear hexapeptide, angiotensin IV (AngIV) have proved to be potent inhibitors of insulin-regulated aminopeptidase (IRAP, oxytocinase, EC 3.4.11.3). Along with higher affinity, macrocycles may also offer better metabolic stability, membrane permeability and selectivity, however predicting the outcome of particular cycle modifications is challenging. Here we describe the development of a series of macrocyclic IRAP inhibitors with either disulphide, olefin metathesis or lactam bridges and variations of ring size and other functionality. The binding mode of these compounds is proposed based on molecular dynamics analysis. Estimation of binding affinities (∆G) and relative binding free energies (∆∆G) with the linear interaction energy (LIE) method and free energy perturbation (FEP) method showed good general agreement with the observed inhibitory potency. Experimental and calculated data highlight the cumulative importance of an intact N-terminal peptide, the specific nature of the macrocycle, the phenolic oxygen and the C-terminal functionality.

  • 227.
    Barrenas, Fredrik
    et al.
    Univ Washington, Dept Microbiol, Seattle, WA 98195 USA..
    Raehetz, Kevin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kristoff, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Agricola, Brian
    Univ Pittsburgh, Sch Med, Dept Microbiol & Mol Genet, Pittsburgh, PA USA..
    Apetrei, Cristian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Agy, Michael B.
    Univ Pittsburgh, Sch Med, Dept Microbiol & Mol Genet, Pittsburgh, PA USA..
    Carter, Victoria
    Univ Washington, Washington Natl Primate Res Ctr, Seattle, WA 98195 USA..
    Flanary, Leon
    Univ Pittsburgh, Sch Med, Dept Microbiol & Mol Genet, Pittsburgh, PA USA..
    Green, Richard R.
    Univ Washington, Washington Natl Primate Res Ctr, Seattle, WA 98195 USA..
    Ma, DongZhu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    McLain, Randy
    Univ Pittsburgh, Sch Med, Dept Microbiol & Mol Genet, Pittsburgh, PA USA..
    Murnane, Robert
    Univ Pittsburgh, Sch Med, Dept Microbiol & Mol Genet, Pittsburgh, PA USA..
    Peng, Xinxia
    Univ Washington, Washington Natl Primate Res Ctr, Seattle, WA 98195 USA..
    Richter, George H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Thomas, Matthew J.
    Univ Washington, Washington Natl Primate Res Ctr, Seattle, WA 98195 USA..
    Trichel, Anita
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Weiss, Jeffrey M.
    Univ Washington, Washington Natl Primate Res Ctr, Seattle, WA 98195 USA..
    Anderson, David M.
    Univ Pittsburgh, Sch Med, Dept Microbiol & Mol Genet, Pittsburgh, PA USA..
    Pandrea, Ivona
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Katze, Michael G.
    Univ Washington, Dept Microbiol, Seattle, WA 98195 USA.;Univ Washington, Washington Natl Primate Res Ctr, Seattle, WA 98195 USA..
    Rhesus Macaques and African Green Monkeys Exhibit Striking Differences in Extracellular Matrix and Cell Adhesion Gene Expression During the Eclipse Phase of Siv Infection2015In: Journal of medical primatology, ISSN 0047-2565, E-ISSN 1600-0684, Vol. 44, no 5, p. 343-343Article in journal (Other academic)
  • 228.
    Barrenäs, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Green, Richard R.
    Thomas, Matthew J.
    Law, G. Lynn
    Proll, Sean C.
    Engelmann, Flora
    Messaoudi, Ilhem
    Marzi, Andrea
    Feldmann, Heinz
    Katze, Michael G.
    Next-Generation Sequencing Reveals a Controlled Immune Response to Zaire Ebola Virus Challenge in Cynomolgus Macaques Immunized with Vesicular Stomatitis Virus Expressing Zaire Ebola Virus Glycoprotein (VSV Delta G/EBOVgp)2015In: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 22, no 3, p. 354-356Article in journal (Refereed)
    Abstract [en]

    Vesicular stomatitis virus expressing Zaire Ebola virus (EBOV) glycoprotein (VSV Delta G/EBOVgp) could be used as a vaccine to meet the 2014 Ebola virus outbreak. To characterize the host response to this vaccine, we used mRNA sequencing to analyze peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques after VSV Delta G/EBOVgp immunization and subsequent EBOV challenge. We found a controlled transcriptional response that transitioned to immune regulation as the EBOV was cleared. This observation supports the safety of the vaccine.

  • 229.
    Barrenäs, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Palermo, Robert E.
    Agricola, Brian
    Agy, Michael B.
    Aicher, Lauri
    Carter, Victoria
    Flanary, Leon
    Green, Richard R.
    McLain, Randy
    Li, Qingsheng
    Lu, Wuxun
    Murnane, Robert
    Peng, Xinxia
    Thomas, Matthew J.
    Weiss, Jeffrey M.
    Anderson, David M.
    Katze, Michael G.
    Deep Transcriptional Sequencing of Mucosal Challenge Compartment from Rhesus Macaques Acutely Infected with Simian Immunodeficiency Virus Implicates Loss of Cell Adhesion Preceding Immune Activation2014In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 88, no 14, p. 7962-7972Article in journal (Refereed)
    Abstract [en]

    Pathology resulting from human immunodeficiency virus (HIV) infection is driven by protracted inflammation; the primary loss of CD4(+) T cells is caused by activation-driven apoptosis. Recent studies of nonhuman primates (NHPs) have suggested that during the acute phase of infection, antiviral mucosal immunity restricts viral replication in the primary infection compartment. These studies imply that HIV achieves systemic infection as a consequence of a failure in host antiviral immunity. Here, we used high-dose intrarectal inoculation of rhesus macaques with simian immunodeficiency virus (SIV) SIVmac251 to examine how the mucosal immune system is overcome by SIV during acute infection. The host response in rectal mucosa was characterized by deep mRNA sequencing (mRNA-seq) at 3 and 12 days postinoculation (dpi) in 4 animals for each time point. While we observed a strong host transcriptional response at 3 dpi, functions relating to antiviral immunity were absent. Instead, we observed a significant number of differentially expressed genes relating to cell adhesion and reorganization of the cytoskeleton. We also observed downregulation of genes encoding members of the claudin family of cell adhesion molecules, which are coexpressed with genes associated with pathology in the colorectal mucosa, and a large number of noncoding transcripts. In contrast, at 12 dpi the differentially expressed genes were enriched in those involved with immune system functions, in particular, functions relating to T cells, B cells, and NK cells. Our findings indicate that host responses that negatively affect mucosal integrity occur before inflammation. Consequently, when inflammation is activated at peak viremia, mucosal integrity is already compromised, potentially enabling rapid tissue damage, driving further inflammation. IMPORTANCE The HIV pandemic is one of the major threats to human health, causing over a million deaths per year. Recent studies have suggested that mucosal antiviral immune responses play an important role in preventing systemic infection after exposure to the virus. Yet, despite their potential role in decreasing transmission rates between individuals, these antiviral mechanisms are poorly understood. Here, we carried out the first deep mRNA sequencing analysis of mucosal host responses in the primary infection compartment during acute SIV infection. We found that during acute infection, a significant host response was mounted in the mucosa before inflammation was triggered. Our analysis indicated that the response has a detrimental effect on tissue integrity, causing increased permeability, tissue damage, and recruitment of SIV target cells. These results emphasize the importance of mucosal host responses preceding immune activation in preventing systemic SIV infection.

  • 230.
    Barrio, Alvaro Martinez
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ekerljung, Marie
    Jern, Patric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Benachenhou, Farid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Sperber, Göran O.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Bongcam-Rudloff, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Andersson, Göran
    The First Sequenced Carnivore Genome Shows Complex Host-Endogenous Retrovirus Relationships2011In: PLOS ONE, ISSN 1932-6203, Vol. 6, no 5, p. e19832-Article in journal (Refereed)
    Abstract [en]

    Host-retrovirus interactions influence the genomic landscape and have contributed substantially to mammalian genome evolution. To gain further insights, we analyzed a female boxer (Canis familiaris) genome for complexity and integration pattern of canine endogenous retroviruses (CfERV). Intriguingly, the first such in-depth analysis of a carnivore species identified 407 CfERV proviruses that represent only 0.15% of the dog genome. In comparison, the same detection criteria identified about six times more HERV proviruses in the human genome that has been estimated to contain a total of 8% retroviral DNA including solitary LTRs. These observed differences in man and dog are likely due to different mechanisms to purge, restrict and protect their genomes against retroviruses. A novel group of gammaretrovirus-like CfERV with high similarity to HERV-Fc1 was found to have potential for active retrotransposition and possibly lateral transmissions between dog and human as a result of close interactions during at least 10.000 years. The CfERV integration landscape showed a non-uniform intra-and inter-chromosomal distribution. Like in other species, different densities of ERVs were observed. Some chromosomal regions were essentially devoid of CfERVs whereas other regions had large numbers of integrations in agreement with distinct selective pressures at different loci. Most CfERVs were integrated in antisense orientation within 100 kb from annotated protein-coding genes. This integration pattern provides evidence for selection against CfERVs in sense orientation relative to chromosomal genes. In conclusion, this ERV analysis of the first carnivorous species supports the notion that different mammals interact distinctively with endogenous retroviruses and suggests that retroviral lateral transmissions between dog and human may have occurred.

  • 231.
    Barrio, Alvaro Martinez
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Eriksson, Oskar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Badhai, Jitendra
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Fröjmark, Anne-Sophie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Bongcam-Rudloff, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Dahl, Niklas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Schuster, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Targeted Resequencing and Analysis of the Diamond-Blackfan Anemia Disease Locus RPS192009In: PLoS ONE, ISSN 1932-6203, Vol. 4, no 7, p. e6172-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The Ribosomal protein S19 gene locus (RPS19) has been linked to two kinds of red cell aplasia, Diamond-Blackfan Anemia (DBA) and Transient Erythroblastopenia in Childhood (TEC). Mutations in RPS19 coding sequences have been found in 25% of DBA patients, but not in TEC patients. It has been suggested that non-coding RPS19 sequence variants contribute to the considerable clinical variability in red cell aplasia. We therefore aimed at identifying non-coding variations associated with DBA or TEC phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: We targeted a region of 19'980 bp encompassing the RPS19 gene in a cohort of 89 DBA and TEC patients for resequencing. We provide here a catalog of the considerable, previously unrecognized degree of variation in this region. We identified 73 variations (65 SNPs, 8 indels) that all are located outside of the RPS19 open reading frame, and of which 67.1% are classified as novel. We hypothesize that specific alleles in non-coding regions of RPS19 could alter the binding of regulatory proteins or transcription factors. Therefore, we carried out an extensive analysis to identify transcription factor binding sites (TFBS). A series of putative interaction sites coincide with detected variants. Sixteen of the corresponding transcription factors are of particular interest, as they are housekeeping genes or show a direct link to hematopoiesis, tumorigenesis or leukemia (e.g. GATA-1/2, PU.1, MZF-1). CONCLUSIONS: Specific alleles at predicted TFBSs may alter the expression of RPS19, modify an important interaction between transcription factors with overlapping TFBS or remove an important stimulus for hematopoiesis. We suggest that the detected interactions are of importance for hematopoiesis and could provide new insights into individual response to treatment.

  • 232.
    Barrio, Alvaro Martínez
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Lagercrantz, Erik
    Sperber, Göran O.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Bongcam-Rudloff, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Annotation and visualization of endogenous retroviral sequences using the Distributed Annotation System (DAS) and eBioX2009In: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 10 Suppl. 6, p. S18-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The Distributed Annotation System (DAS) is a widely used network protocol for sharing biological information. The distributed aspects of the protocol enable the use of various reference and annotation servers for connecting biological sequence data to pertinent annotations in order to depict an integrated view of the data for the final user. RESULTS: An annotation server has been devised to provide information about the endogenous retroviruses detected and annotated by a specialized in silico tool called RetroTector. We describe the procedure to implement the DAS 1.5 protocol commands necessary for constructing the DAS annotation server. We use our server to exemplify those steps. Data distribution is kept separated from visualization which is carried out by eBioX, an easy to use open source program incorporating multiple bioinformatics utilities. Some well characterized endogenous retroviruses are shown in two different DAS clients. A rapid analysis of areas free from retroviral insertions could be facilitated by our annotations. CONCLUSION: The DAS protocol has shown to be advantageous in the distribution of endogenous retrovirus data. The distributed nature of the protocol is also found to aid in combining annotation and visualization along a genome in order to enhance the understanding of ERV contribution to its evolution. Reference and annotation servers are conjointly used by eBioX to provide visualization of ERV annotations as well as other data sources. Our DAS data source can be found in the central public DAS service repository, http://www.dasregistry.org, or at http://loka.bmc.uu.se/das/sources.

  • 233.
    Barrozo, Alexandre
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Promiscuity and Selectivity in Phosphoryl Transferases2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Phosphoryl transfers are essential chemical reactions in key life processes, including energy production, signal transduction and protein synthesis. They are known for having extremely low reaction rates in aqueous solution, reaching the scale of millions of years. In order to make life possible, enzymes that catalyse phosphoryl transfer, phosphoryl transferases, have evolved to be tremendously proficient catalysts, increasing reaction rates to the millisecond timescale.

    Due to the nature of the electronic structure of phosphorus atoms, understanding how hydrolysis of phosphate esters occurs is a complex task. Experimental studies on the hydrolysis of phosphate monoesters with acidic leaving groups suggest a concerted mechanism with a loose, metaphosphate-like transition state. Theoretical studies have suggested two possible concerted pathways, either with loose or tight transition state geometries, plus the possibility of a stepwise mechanism with the formation of a phosphorane intermediate. Different pathways were shown to be energetically preferable depending on the acidity of the leaving group. Here we performed computational studies to revisit how this mechanistic shift occurs along a series of aryl phosphate monoesters, suggesting possible factors leading to such change.

    The fact that distinct pathways can occur in solution could mean that the same is possible for an enzyme active site. We performed simulations on the catalytic activity of β-phosphoglucomutase, suggesting that it is possible for two mechanisms to occur at the same time for the phosphoryl transfer.

    Curiously, several phosphoryl transferases were shown to be able to catalyse not only phosphate ester hydrolysis, but also the cleavage of other compounds. We modeled the catalytic mechanism of two highly promiscuous members of the alkaline phosphatase superfamily. Our model reproduces key experimental observables and shows that these enzymes are electrostatically flexible, employing the same set of residues to enhance the rates of different reactions, with different electrostatic contributions per residue.

    List of papers
    1. Evaluation and Characterisation of Mechanistic Alternatives for beta-Phosphoglucomutase
    Open this publication in new window or tab >>Evaluation and Characterisation of Mechanistic Alternatives for beta-Phosphoglucomutase
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-278943 (URN)
    Available from: 2016-02-26 Created: 2016-02-26 Last updated: 2016-04-12
    2. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Open this publication in new window or tab >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
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    2014 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 16, p. 4351-4362Article in journal (Refereed) Published
    Abstract [en]

    The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

    National Category
    Physical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
    Funder
    Swedish National Infrastructure for Computing (SNIC), 2013/26-1
    Available from: 2014-06-23 Created: 2014-06-04 Last updated: 2018-12-03Bibliographically approved
    3. Mechanistic Shifts Along the Linear Free Energy Relationship for Aryl Phosphate Monoester Hydrolysis
    Open this publication in new window or tab >>Mechanistic Shifts Along the Linear Free Energy Relationship for Aryl Phosphate Monoester Hydrolysis
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Theoretical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-278945 (URN)
    Available from: 2016-02-26 Created: 2016-02-26 Last updated: 2016-04-12
    4. Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily
    Open this publication in new window or tab >>Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily
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    2015 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, no 28, p. 9061-9076Article in journal (Refereed) Published
    Abstract [en]

    It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-260856 (URN)10.1021/jacs.5b03945 (DOI)000358556200033 ()26091851 (PubMedID)
    Funder
    Swedish Research Council, 2010-5026EU, FP7, Seventh Framework Programme, 306474Swedish National Infrastructure for Computing (SNIC), 25/2-10
    Note

    De 2 första författarna delar förstaförfattarskapet.

    Available from: 2015-08-26 Created: 2015-08-25 Last updated: 2017-12-04Bibliographically approved
  • 234.
    Barrozo, Alexandre
    et al.
    Univ Southern Calif, Dept Chem, Los Angeles, CA 90089 USA..
    Blaha-Nelson, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Williams, Nicholas H.
    Univ Sheffield, Dept Chem, Sheffield S3 7HF, S Yorkshire, England..
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    The effect of magnesium ions on triphosphate hydrolysis2017In: Pure and Applied Chemistry, ISSN 0033-4545, E-ISSN 1365-3075, Vol. 89, no 6, p. 715-727Article in journal (Refereed)
    Abstract [en]

    The role of metal ions in catalyzing phosphate ester hydrolysis has been the subject of much debate, both in terms of whether they change the transition state structure or mechanistic pathway. Understanding the impact of metal ions on these biologically critical reactions is central to improving our understanding of the role of metal ions in the numerous enzymes that facilitate them. In the present study, we have performed density functional theory studies of the mechanisms of methyl triphosphate and acetyl phosphate hydrolysis in aqueous solution to explore the competition between solvent-and substrate-assisted pathways, and examined the impact of Mg2+ on the energetics and transition state geometries. In both cases, we observe a clear preference for a more dissociative solvent-assisted transition state, which is not significantly changed by coordination of Mg2+. The effect of Mg2+ on the transition state geometries for the two pathways is minimal. While our calculations cannot rule out a substrate-assisted pathway as a possible solution for biological phosphate hydrolysis, they demonstrate that a significantly higher energy barrier needs to be overcome in the enzymatic reaction for this to be an energetically viable reaction pathway.

  • 235.
    Barrozo, Alexandre
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Borstnar, Rok
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Marloie, Gaël
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Kamerlin, Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Computational Protein Engineering: Bridging the Gap between Rational Design and Laboratory Evolution2012In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 13, no 10, p. 12428-12460Article, review/survey (Refereed)
    Abstract [en]

    Enzymes are tremendously proficient catalysts, which can be used as extracellular catalysts for a whole host of processes, from chemical synthesis to the generation of novel biofuels. For them to be more amenable to the needs of biotechnology, however, it is often necessary to be able to manipulate their physico-chemical properties in an efficient and streamlined manner, and, ideally, to be able to train them to catalyze completely new reactions. Recent years have seen an explosion of interest in different approaches to achieve this, both in the laboratory, and in silico. There remains, however, a gap between current approaches to computational enzyme design, which have primarily focused on the early stages of the design process, and laboratory evolution, which is an extremely powerful tool for enzyme redesign, but will always be limited by the vastness of sequence space combined with the low frequency for desirable mutations. This review discusses different approaches towards computational enzyme design and demonstrates how combining newly developed screening approaches that can rapidly predict potential mutation “hotspots” with approaches that can quantitatively and reliably dissect the catalytic step can bridge the gap that currently exists between computational enzyme design and laboratory evolution studies.

  • 236.
    Barrozo, Alexandre
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Duarte, Fernanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Carvalho, Alexandra T. P.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily2015In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, no 28, p. 9061-9076Article in journal (Refereed)
    Abstract [en]

    It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.

  • 237.
    Barrozo, Alexandre
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Esguerra, Mauricio
    Marloie, Gael
    Florian, Jan
    Williams, Nicholas
    Kamerlin, Shina
    Evaluation and Characterisation of Mechanistic Alternatives for beta-PhosphoglucomutaseManuscript (preprint) (Other academic)
  • 238.
    Barrozo, Alexandre H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Harnessing Promiscuity Patterns to Map Evolution in the Alkaline Phosphatase Superfamily2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 232A-232AArticle in journal (Other academic)
  • 239.
    Barrozo, Alexandre H.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Carvalho, Alexandra Pires
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Understanding Functional Evolution in the Alkaline Phosphatase Superfamily2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 675A-675AArticle in journal (Other academic)
    Abstract [en]

    Over the past 40 years, it has been demonstrated that many enzymes are capable of promiscuous catalytic activities, facilitating the turnover of more than one chemically distinct substrate. This has been argued to play an important role in enzyme evolution, with highly promiscuous progenitor enzymes evolving under evolutionary pressure to modern day specialists, while still retaining some level of their former promiscuous activities1. This theory has been extensively tested by different experiments using in vitro evolution2. The alkaline phosphatase superfamily members provide a particularly attractive showcase for studying enzyme promiscuity, as they often show reciprocal promiscuity, in that the native reaction for one member is often a side-reaction for another3. While deceptively similar, their catalyzed reactions (cleavage of P-O and S-O bonds) proceed via distinct transition states and protonation requirements4,5. We present detailed computational studies of the promiscuous catalytic activity of three evolutionarily related members: the arylsulfatase from Pseudomonas aeruginosa6, and the phosphonate monoester hydrolases from Burkholderia caryophili7and Rhizobium leguminosarum8. By tracking their structural and electrostatic features, and comparing to other known members of the superfamily, we provide an atomic-level map for functional evolution within this superfamily.

  • 240.
    Barrozo, Alexandre
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Brandao, Tiago
    Hengge, Alvan
    Phosphoryl and Sulfuryl Transfer2016In: Reference Module in Chemistry, Molecular Sciences and Chemical EngineeringArticle in journal (Refereed)
  • 241.
    Barrozo, Alexandre
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Liao, Qinghua
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Esguerra, Mauricio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Marloie, Gael
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Florian, Jan
    Loyola Univ Chicago, Dept Chem & Biochem, Chicago, IL 60660 USA..
    Williams, Nicholas H.
    Univ Sheffield, Dept Chem, Sheffield S3 7HF, S Yorkshire, England..
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Computer simulations of the catalytic mechanism of wild-type and mutant beta-phosphoglucomutase2018In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 16, no 12, p. 2060-2073Article in journal (Refereed)
    Abstract [en]

    beta-Phosphoglucomutase (beta-PGM) has served as an important model system for understanding biological phosphoryl transfer. This enzyme catalyzes the isomerization of beta-glucose-1-phosphate to -glucose-6-phosphate in a two-step process proceeding via a bisphosphate intermediate. The conventionally accepted mechanism is that both steps are concerted processes involving acid-base catalysis from a nearby aspartate (D10) side chain. This argument is supported by the observation that mutation of D10 leaves the enzyme with no detectable activity. However, computational studies have suggested that a substrate-assisted mechanism is viable for many phosphotransferases. Therefore, we carried out empirical valence bond (EVB) simulations to address the plausibility of this mechanistic alternative, including its role in the abolished catalytic activity of the D10S, D10C and D10N point mutants of beta-PGM. In addition, we considered both of these mechanisms when performing EVB calculations of the catalysis of the wild type (WT), H20A, H20Q, T16P, K76A, D170A and E169A/D170A protein variants. Our calculated activation free energies confirm that D10 is likely to serve as the general base/acid for the reaction catalyzed by the WT enzyme and all its variants, in which D10 is not chemically altered. Our calculations also suggest that D10 plays a dual role in structural organization and maintaining electrostatic balance in the active site. The correct positioning of this residue in a catalytically competent conformation is provided by a functionally important conformational change in this enzyme and by the extensive network of H-bonding interactions that appear to be exquisitely preorganized for the transition state stabilization.

  • 242.
    Bartoschek, Michael
    et al.
    Lund Univ, Dept Lab Med, Div Translat Canc Res, BioCARE, S-22381 Lund, Sweden.
    Oskolkov, Nikolay
    Lund Univ, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Dept Biol, Solvegatan 35, S-22362 Lund, Sweden.
    Bocci, Matteo
    Lund Univ, Dept Lab Med, Div Translat Canc Res, BioCARE, S-22381 Lund, Sweden.
    Lovrot, John
    Karolinska Inst, Dept Oncol & Pathol, Karolinska Univ Sjukhuset Z1 01, S-17176 Stockholm, Sweden.
    Larsson, Christer
    Lund Univ, Dept Lab Med, Div Translat Canc Res, BioCARE, S-22381 Lund, Sweden.
    Sommarin, Mikael
    Lund Univ, Lund Stem Cell Ctr, Div Mol Hematol, BMC B12, S-22184 Lund, Sweden.
    Madsen, Chris D.
    Lund Univ, Dept Lab Med, Div Translat Canc Res, BioCARE, S-22381 Lund, Sweden.
    Lindgren, David
    Lund Univ, Dept Lab Med, Div Translat Canc Res, BioCARE, S-22381 Lund, Sweden.
    Pekar, Gyula
    Lund Univ, Dept Clin Sci, Div Oncol & Pathol, Skane Univ Hosp, S-22185 Lund, Sweden.
    Karlsson, Goran
    Lund Univ, Lund Stem Cell Ctr, Div Mol Hematol, BMC B12, S-22184 Lund, Sweden.
    Ringner, Markus
    Lund Univ, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Dept Biol, Solvegatan 35, S-22362 Lund, Sweden.
    Bergh, Jonas
    Karolinska Inst, Dept Oncol & Pathol, Karolinska Univ Sjukhuset Z1 01, S-17176 Stockholm, Sweden.
    Björklund, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pietras, Kristian
    Lund Univ, Dept Lab Med, Div Translat Canc Res, BioCARE, S-22381 Lund, Sweden.
    Spatially and functionally distinct subclasses of breast cancer-associated fibroblasts revealed by single cell RNA sequencing2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 5150Article in journal (Refereed)
    Abstract [en]

    Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment, although their origin and roles in shaping disease initiation, progression and treatment response remain unclear due to significant heterogeneity. Here, following a negative selection strategy combined with single-cell RNA sequencing of 768 transcriptomes of mesenchymal cells from a genetically engineered mouse model of breast cancer, we define three distinct subpopulations of CAFs. Validation at the transcriptional and protein level in several experimental models of cancer and human tumors reveal spatial separation of the CAF subclasses attributable to different origins, including the peri-vascular niche, the mammary fat pad and the transformed epithelium. Gene profiles for each CAF subtype correlate to distinctive functional programs and hold independent prognostic capability in clinical cohorts by association to metastatic disease. In conclusion, the improved resolution of the widely defined CAF population opens the possibility for biomarker-driven development of drugs for precision targeting of CAFs.

  • 243. Barty, Anton
    et al.
    Boutet, Sebastien
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bogan, Michael J.
    Hau-Riege, Stefan
    Marchesini, Stefano
    Sokolowski-Tinten, Klaus
    Stojanovic, Nikola
    Tobey, Ra'Anan
    Ehrke, Henri
    Cavalleri, Andrea
    Duesterer, Stefan
    Frank, Matthias
    Bajt, Sasa
    Woods, Bruce W.
    Seibert, M. Marvin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Treusch, Rolf
    Chapman, Henry N.
    Ultrafast single-shot diffraction imaging of nanoscale dynamics2008In: Nature Photonics, ISSN 1749-4885, Vol. 2, no 7, p. 415-419Article in journal (Refereed)
    Abstract [en]

    The transient nanoscale dynamics of materials on femtosecond to picosecond timescales is of great interest in the study of condensed phase dynamics such as crack formation, phase separation and nucleation, and rapid fluctuations in the liquid state or in biologically relevant environments. The ability to take images in a single shot is the key to studying non-repetitive behaviour mechanisms, a capability that is of great importance in many of these problems. Using coherent diffraction imaging with femtosecond X-ray free-electron-laser pulses we capture time-series snapshots of a solid as it evolves on the ultrafast timescale. Artificial structures imprinted on a Si3N4 window are excited with an optical laser and undergo laser ablation, which is imaged with a spatial resolution of 50 nm and a temporal resolution of 10 ps. By using the shortest available free-electron-laser wavelengths(1) and proven synchronization methods(2) this technique could be extended to spatial resolutions of a few nanometres and temporal resolutions of a few tens of femtoseconds. This experiment opens the door to a new regime of time-resolved experiments in mesoscopic dynamics.

  • 244. Barty, Anton
    et al.
    Caleman, Carl
    Aquila, Andrew
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Lomb, Lukas
    White, Thomas A.
    Andreasson, Jakob
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Arnlund, David
    Bajt, Sasa
    Barends, Thomas R. M.
    Barthelmess, Miriam
    Bogan, Michael J.
    Bostedt, Christoph
    Bozek, John D.
    Coffee, Ryan
    Coppola, Nicola
    Davidsson, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry.
    DePonte, Daniel P.
    Doak, R. Bruce
    Ekeberg, Tomas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Elser, Veit
    Epp, Sascha W.
    Erk, Benjamin
    Fleckenstein, Holger
    Foucar, Lutz
    Fromme, Petra
    Graafsma, Heinz
    Gumprecht, Lars
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hampton, Christina Y.
    Hartmann, Robert
    Hartmann, Andreas
    Hauser, Guenter
    Hirsemann, Helmut
    Holl, Peter
    Hunter, Mark S.
    Johansson, Linda
    Kassemeyer, Stephan
    Kimmel, Nils
    Kirian, Richard A.
    Liang, Mengning
    Maia, Filipe R. N. C.
    Malmerberg, Erik
    Marchesini, Stefano
    Martin, Andrew V.
    Nass, Karol
    Neutze, Richard
    Reich, Christian
    Rolles, Daniel
    Rudek, Benedikt
    Rudenko, Artem
    Scott, Howard
    Schlichting, Ilme
    Schulz, Joachim
    Seibert, M. Marvin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Shoeman, Robert L.
    Sierra, Raymond G.
    Soltau, Heike
    Spence, John C. H.
    Stellato, Francesco
    Stern, Stephan
    Strueder, Lothar
    Ullrich, Joachim
    Wang, X.
    Weidenspointner, Georg
    Weierstall, Uwe
    Wunderer, Cornelia B.
    Chapman, Henry N.
    Self-terminating diffraction gates femtosecond X-ray nanocrystallography measurements2012In: Nature Photonics, ISSN 1749-4885, E-ISSN 1749-4893, Vol. 6, no 1, p. 35-40Article in journal (Refereed)
    Abstract [en]

    X-ray free-electron lasers have enabled new approaches to the structural determination of protein crystals that are too small or radiation-sensitive for conventional analysis(1). For sufficiently short pulses, diffraction is collected before significant changes occur to the sample, and it has been predicted that pulses as short as 10 fs may be required to acquire atomic-resolution structural information(1-4). Here, we describe a mechanism unique to ultrafast, ultra-intense X-ray experiments that allows structural information to be collected from crystalline samples using high radiation doses without the requirement for the pulse to terminate before the onset of sample damage. Instead, the diffracted X-rays are gated by a rapid loss of crystalline periodicity, producing apparent pulse lengths significantly shorter than the duration of the incident pulse. The shortest apparent pulse lengths occur at the highest resolution, and our measurements indicate that current X-ray free-electron laser technology(5) should enable structural determination from submicrometre protein crystals with atomic resolution.

  • 245. Barty, Anton
    et al.
    Kirian, Richard A.
    Maia, Filipe R. N. C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hantke, Max
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Yoon, Chun Hong
    White, Thomas A.
    Chapman, Henry
    Cheetah: software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data2014In: Journal of applied crystallography, ISSN 0021-8898, E-ISSN 1600-5767, Vol. 47, p. 1118-1131Article in journal (Refereed)
    Abstract [en]

    The emerging technique of serial X-ray diffraction, in which diffraction data are collected from samples flowing across a pulsed X-ray source at repetition rates of 100 Hz or higher, has necessitated the development of new software in order to handle the large data volumes produced. Sorting of data according to different criteria and rapid filtering of events to retain only diffraction patterns of interest results in significant reductions in data volume, thereby simplifying subsequent data analysis and management tasks. Meanwhile the generation of reduced data in the form of virtual powder patterns, radial stacks, histograms and other meta data creates data set summaries for analysis and overall experiment evaluation. Rapid data reduction early in the analysis pipeline is proving to be an essential first step in serial imaging experiments, prompting the authors to make the tool described in this article available to the general community. Originally developed for experiments at X-ray free-electron lasers, the software is based on a modular facility-independent library to promote portability between different experiments and is available under version 3 or later of the GNU General Public License.

  • 246.
    Barz, Bogdan
    et al.
    Forschungszentrum Jülich GmbH, Institute of Complex Systems: Structural Biochemistry (ICS-6), Jülich; Heinrich Heine University Düsseldorf, Institute of Theoretical and Computational Chemistry, Düsseldorf.
    Liao, Qinghua
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Forschungszentrum Jülich GmbH, Institute of Complex Systems: Structural Biochemistry (ICS-6), Jülich.
    Strodel, Birgit
    Forschungszentrum Jülich GmbH, Institute of Complex Systems: Structural Biochemistry (ICS-6), Jülich; Heinrich Heine University Düsseldorf, Institute of Theoretical and Computational Chemistry, Düsseldorf.
    Pathways of Amyloid-β Aggregation Depend on Oligomer Shape2018In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, no 1, p. 319-327Article in journal (Refereed)
    Abstract [en]

    One of the main research topics related to Alzheimer’s disease is the aggregation of the amyloid-β peptide, which was shown to follow different pathways for the two major alloforms of the peptide, Aβ40 and the more toxic Aβ42. Experimental studies emphasized that oligomers of specific sizes appear in the early aggregation process in different quantities and might be the key toxic agents for each of the two alloforms. We use transition networks derived from all-atom molecular dynamics simulations to show that the oligomers leading to the type of oligomer distributions observed in experiments originate from compact conformations. Extended oligomers, on the other hand, contribute more to the production of larger aggregates thus driving the aggregation process. We further demonstrate that differences in the aggregation pathways of the two Aβ alloforms occur as early as during the dimer stage. The higher solvent-exposure of hydrophobic residues in Aβ42 oligomers contributes to the different aggregation pathways of both alloforms and also to the increased cytotoxicity of Aβ42.

  • 247.
    Bashardanesh, Zahedeh
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Lötstedt, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Numerical Analysis.
    Efficient Green's function reaction dynamics (GFRD) simulations for diffusion-limited, reversible reactions2018In: Journal of Computational Physics, ISSN 0021-9991, E-ISSN 1090-2716, Vol. 357, p. 78-99Article in journal (Refereed)
  • 248.
    Bashardanesh, Zahedeh
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Impact of Dispersion Coefficient on Simulations of Proteins and Organic Liquids2018In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 122, no 33, p. 8018-8027Article in journal (Refereed)
    Abstract [en]

    In the context of studies of proteins under crowding conditions, it was found that there is a tendency of simulated proteins to coagulate in a seemingly unphysical manner. This points to an imbalance in the protein-protein or protein-water interactions. One way to resolve this is to strengthen the protein-water Lennard-Jones interactions. However, it has also been suggested that dispersion interactions may have been systematically overestimated in force fields due to parameterization with a short cutoff. Here, we test this proposition by performing simulations of liquids and of proteins in solution with systematically reduced C-6 (dispersion constant in a 12-6 Lennard-Jones potential) and evaluate the properties. We find that simulations of liquids with either a dispersion correction or explicit long-range Lennard-Jones interactions need little or no correction to the dispersion constant to reproduce the experimental density. For simulations of proteins, a significant reduction in the dispersion constant is needed to reduce the coagulation, however. Because the protein- and liquid force fields share atom types, at least to some extent, another solution for the coagulation problem may be needed, either through including explicit polarization or through strengthening protein-water interactions.

  • 249.
    Bashardanesh, Zahedeh
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Zhang, Haiyang
    University of Science and Technology Beijing.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Rotational and translational diffusion of proteins as a function of concentrationManuscript (preprint) (Other academic)
  • 250. Basile, Maria
    et al.
    Lin, Ridwan
    Kabbani, Nadine
    Karpa, Kelly
    Kilimann, Manfred W.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Simpson, Ian
    Kester, Mark
    Paralemmin interacts with D3 dopamine receptors: implications for membrane localization and cAMP signaling2006In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 446, no 1, p. 60-68Article in journal (Refereed)
    Abstract [en]

    Paralemmin is a novel lipid-anchored protein, which is highly expressed in neuronal plasma membranes. In this study, we demonstrate that paralemmin specifically interacts with the third intracellular loop of the D3 dopamine receptor. Utilizing co-immunoprecipitation and glutathione-S-transferase (GST) pulldown strategies, we demonstrate that paralemmin interacts exclusively with D3, but not D2 or D4 dopamine receptors or β-adrenergic receptors. Immunocytochemistry demonstrated co-localization of paralemmin and D3 receptor in vivo in hippocampus and cerebellum and in vitro in glial and neuronal cultures. Deletion mutational analysis indicates that amino acids 154–230 of paralemmin strongly interacted with amino acids 211–227 and 281–330 of the third intracellular loop of D3 receptor. The consequences of these interactions were investigated by co-expression in HEK293 cells. Cell surface biotinylation experiments demonstrate that paralemmin decreased D3 receptor concentration at the plasma membrane. Consistent with this observation, paralemmin expression decreased dopamine-stimulated adenylate cyclase activity. However, paralemmin also decreased basal, isoproterenol and forskolin-stimulated adenylate cyclase activity, suggesting a more general cellular function for paralemmin. Taken together, paralemmin has been implicated as a potent modulator of cellular cAMP signaling within the brain.

2345678 201 - 250 of 3218
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