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  • 151.
    Antoun, Ayman
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pavlov, Michael
    Ehrenberg, Måns
    Ribosomal recycling by the interplay of RRF and three conformations of EFGManuskript (Övrigt vetenskapligt)
  • 152. Antoun, Ayman
    et al.
    Pavlov, Michael
    Lovmar, Martin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Ehrenberg, Måns
    Accuracy of initiator tRNA selection in initiation of eubacterial protein synthesisManuskript (Övrigt vetenskapligt)
  • 153.
    Antoun, Ayman
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pavlov, Michael
    Lovmar, Martin
    Ehrenberg, Måns
    Accuracy of initiator tRNA selection in initiation of eubacterial protein synthesisManuskript (Övrigt vetenskapligt)
  • 154.
    Antoun, Ayman
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pavlov, Michael
    Lovmar, Martin
    Ehrenberg, Måns
    Interplay of initiation factors and initiator tRNA in initiation of eubacterial protein synthesisManuskript (Övrigt vetenskapligt)
  • 155. Antoun, Ayman
    et al.
    Pavlov, Michael
    Lovmar, Martin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Ehrenberg, Måns
    Tuning of initiation of eubacterial protein synthesis by initiation factorsManuskript (Övrigt vetenskapligt)
  • 156. Antoun, Ayman
    et al.
    Pavlov, Michael
    Lovmar, Martin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Ehrenberg, Måns
    Why is initiation factor 1 essential for E. coli?Manuskript (Övrigt vetenskapligt)
  • 157.
    Antoun, Ayman
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pavlov, Michael
    Lovmar, Martin
    Ehrenberg, Måns
    Why is initiation factor IF1 essential for E.Coli?Manuskript (Övrigt vetenskapligt)
  • 158.
    Antoun, Ayman
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pavlov, Michael
    Tenson, Tanel
    Ehrenberg, Måns
    Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering2004Ingår i: Biological procedure online, Vol. 6, nr 1, s. 35-54Artikel i tidskrift (Refereegranskat)
  • 159.
    Antoun, Ayman
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Molekylärbiologi.
    Pavlov, Michael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Molekylärbiologi.
    Tenson, Tanel
    Ehrenberg, Måns
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Molekylärbiologi.
    Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering2004Ingår i: Biol Procedure Online, Vol. 6, s. 35–54-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is high-lighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix.

  • 160.
    Antoun, Ayman
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pavlov, Michael Y.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Andersson, Kerstin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Tenson, Tanel
    Ehrenberg, Måns
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis2003Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, nr 20, s. 5593-5601Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2–GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2.

  • 161.
    Antoun, Ayman
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pavlov, Michael Y.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Lovmar, Martin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Ehrenberg, Måns
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis2006Ingår i: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 23, nr 2, s. 183-193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    During initiation of bacterial protein synthesis, messenger RNA and fMet-tRNA(fMet) bind to the 30S ribosomal subunit together with initiation factors IF1, IF2, and IF3. Docking of the 30S preinitiation complex to the 50S ribosomal subunit results in a peptidyl-transfer competent 70S ribosome. Initiation with an elongator tRNA may lead to frameshift and an aberrant N-terminal sequence in the nascent protein. We show how the occurrence of initiation errors is minimized by (1) recognition of the formyl group by the synergistic action of IF2 and IF1, (2) uniform destabilization of the binding of all tRNAs to the 30S subunit by IF3, and (3) an optimal distance between the Shine-Dalgarno sequence and the initiator codon. We suggest why IF1 is essential for E. coli, discuss the role of the G-C base pairs in the anticodon stem of some tRNAs, and clarify gene expression changes with varying IF3 concentration in the living cell.

  • 162.
    Antoun, Ayman
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pavlov, Michael Y
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Lovmar, Martin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Ehrenberg, Måns
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    How initiation factors tune the rate of initiation of protein synthesis in bacteria.2006Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 25, nr 11, s. 2539-50Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The kinetics of initiator transfer RNA ( tRNA) interaction with the messenger RNA ( mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency ( k(cat)/K-M) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.

  • 163. Aponte-Santamaria, Camilo
    et al.
    Hub, Jochen S.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    de Groot, Bert L.
    Dynamics and energetics of solute permeation through the Plasmodium falciparum aquaglyceroporin2010Ingår i: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 12, nr 35, s. 10246-10254Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The aquaglyceroporin from Plasmodium falciparum (PfAQP) is a potential drug target for the treatment of malaria. It efficiently conducts water and other small solutes, and is proposed to intervene in several crucial physiological processes during the parasitic life cycle. Despite the wealth of experimental data available, a dynamical and energetic description at the single-molecule level of the solute permeation through PfAQP has been lacking so far. Here we address this question by using equilibrium and umbrella sampling molecular dynamics simulations. We computed the water osmotic permeability coefficient, the pore geometry and the potential of mean force for the permeation of water, glycerol and urea. Our simulations show that the PfAQP, the human aquaporin 1 (hAQP1) and the Escherichia coli glycerol facilitator (GlpF) have nearly identical water permeabilities. The Arg196 residue at the ar/R region was found to play a crucial role regulating the permeation of water, glycerol and urea. The computed free energy barriers at the ar/R selectivity filter corroborate that PfAQP conducts glycerol at higher rates than urea, and suggest that PfAQP is a more efficient glycerol and urea channel than GlpF. Our results are consistent with a solute permeation mechanism for PfAQP which is similar to the one established for other members of the aquaglyceroporin family. In this mechanism, hydrophobic regions near the NPA motifs are the main water rate limiting barriers, and the replacement of water-arg196 interactions and solute-matching in the hydrophobic pocket at the ar/R region are the main determinants underlying selectivity for the permeation of solutes like glycerol and urea.

  • 164. Apraiz, Itxaso
    et al.
    Mi, Jia
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär cellbiologi.
    Cristobal, Susana
    Identification of proteomic signatures of exposure to marine pollutants in mussels (Mytilus edulis).2006Ingår i: Mol Cell Proteomics, ISSN 1535-9476, Vol. 5, nr 7, s. 1274-85Artikel i tidskrift (Övrigt vetenskapligt)
  • 165. Apraiz, Itxaso
    et al.
    Mi, Jia
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Cristobal, Susana
    Identification of proteomics signatures of exposure to marine pollutants in mussels (Mytilus edulis)2006Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, Vol. 5, nr 7, s. 1274-1285Artikel i tidskrift (Refereegranskat)
  • 166. Aquila, A.
    et al.
    Barty, A.
    Bostedt, C.
    Boutet, S.
    Carini, G.
    dePonte, D.
    Drell, P.
    Doniach, S.
    Downing, K. H.
    Earnest, T.
    Elmlund, H.
    Elser, V.
    Gühr, M.
    Hajdu, Janos
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Hastings, J.
    Hau-Riege, S. P.
    Huang, Z.
    Lattman, E. E.
    Maia, F. R. N. C.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Marchesini, S.
    Ourmazd, A.
    Pellegrini, C.
    Santra, R.
    Schlichting, I.
    Schroer, C.
    Spence, J. C. H.
    Vartanyants, I. A.
    Wakatsuki, S.
    Weis, W. I.
    Williams, G. J.
    The linac coherent light source single particle imaging road map2015Ingår i: Structural Dynamics, Vol. 2, nr 4, artikel-id 041701Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Intense femtosecond x-ray pulses from free-electron laser sources allow the imag-ing of individual particles in a single shot. Early experiments at the Linac CoherentLight Source (LCLS) have led to rapid progress in the field and, so far, coherentdiffractive images have been recorded from biological specimens, aerosols, andquantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLSheld a workshop to discuss the scientific and technical challenges for reaching theultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap towardreaching atomic resolution, 3D imaging at free-electron laser sources.

  • 167. Aquila, Andrew
    et al.
    Hunter, Mark S.
    Doak, R. Bruce
    Kirian, Richard A.
    Fromme, Petra
    White, Thomas A.
    Andreasson, Jakob
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Arnlund, David
    Bajt, Saša
    Barends, Thomas R. M.
    Barthelmess, Miriam
    Bogan, Michael J.
    Bostedt, Christoph
    Bottin, Hervé
    Bozek, John D.
    Caleman, Carl
    Coppola, Nicola
    Davidsson, Jan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Fysikalisk kemi.
    DePonte, Daniel P.
    Elser, Veit
    Epp, Sascha W.
    Erk, Benjamin
    Fleckenstein, Holger
    Foucar, Lutz
    Frank, Matthias
    Fromme, Raimund
    Graafsma, Heinz
    Grotjohann, Ingo
    Gumprecht, Lars
    Hajdu, Janos
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Hampton, Christina Y.
    Hartmann, Andreas
    Hartmann, Robert
    Hau-Riege, Stefan
    Hauser, Günter
    Hirsemann, Helmut
    Holl, Peter
    Holton, James M.
    Hömke, André
    Johansson, Linda
    Kimmel, Nils
    Kassemeyer, Stephan
    Krasniqi, Faton
    Kühnel, Kai-Uwe
    Liang, Mengning
    Lomb, Lukas
    Malmerberg, Erik
    Marchesini, Stefano
    Martin, Andrew V.
    Maia, Filipe R.N.C.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Messerschmidt, Marc
    Nass, Karol
    Reich, Christian
    Neutze, Richard
    Rolles, Daniel
    Rudek, Benedikt
    Rudenko, Artem
    Schlichting, Ilme
    Schmidt, Carlo
    Schmidt, Kevin E.
    Schulz, Joachim
    Seibert, M. Marvin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Soltau, Heike
    Shoeman, Robert L.
    Sierra, Raymond
    Starodub, Dmitri
    Stellato, Francesco
    Stern, Stephan
    Strüder, Lothar
    Timneanu, Nicusor
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Ullrich, Joachim
    Wang, Xiaoyu
    Williams, Garth J.
    Weidenspointner, Georg
    Weierstall, Uwe
    Wunderer, Cornelia
    Barty, Anton
    Spence, John C. H.
    Chapman, Henry N.
    Time-resolved protein nanocrystallography using an X-ray free-electron laser2012Ingår i: Optics Express, ISSN 1094-4087, E-ISSN 1094-4087, Vol. 20, nr 3, s. 2706-2716Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.

  • 168.
    Aqvist, Johan
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Kamerlin, Shina C. L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Conserved Motifs in Different Classes of GTPases Dictate their Specific Modes of Catalysis2016Ingår i: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 6, nr 3, s. 1737-1743Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The GTPase superfamily of enzymes that hydrolyze GTP have a number of conserved sequence regions (the so-called "G-motifs"), and several of the subfamilies also require catalytic activation by specific GTPase-activating proteins. In the translational GTPases involved in protein synthesis, this activating function is instead accomplished by their interaction with the ribosome. Despite these similarities, there are distinct differences regarding some of the amino acid residues making up the GTPase active sites. This raises the question of whether or not the catalytic mechanisms of different types of GTPases are identical. We report herein extensive computer simulations of both the intrinsic GTP hydrolysis reaction of Ras and the considerably faster reaction activated by the interaction with RasGAP. The results of these calculations are compared to earlier simulations of GTP hydrolysis by EF-Tu on the ribosome and show that the favored reaction pathways are strongly dependent on the composition of the active site. By computing Arrhenius plots for the temperature dependence of the calculated free energy profiles, we further show that different mechanistic pathways are associated with distinct differences in activation entropies and enthalpies. The activation parameters are in good agreement with experimental data, and we conclude that calculations of Arrhenius plots from computer simulations can be very useful for dissecting the energetics of enzyme catalysis.

  • 169.
    Aqvist, Johan
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Exceptionally large entropy contributions enable the high rates of GTP hydrolysis on the ribosome2015Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, artikel-id 15817Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Protein synthesis on the ribosome involves hydrolysis of GTP in several key steps of the mRNA translation cycle. These steps are catalyzed by the translational GTPases of which elongation factor Tu (EF-Tu) is the fastest GTPase known. Here, we use extensive computer simulations to explore the origin of its remarkably high catalytic rate on the ribosome and show that it is made possible by a very large positive activation entropy. This entropy term (T Delta S-double dagger) amounts to more than 7 kcal/mol at 25 degrees C. It is further found to be characteristic of the reaction mechanism utilized by the translational, but not other, GTPases and it enables these enzymes to attain hydrolysis rates exceeding 500 s(-1). This entropy driven mechanism likely reflects the very high selection pressure on the speed of protein synthesis, which drives the rate of each individual GTPase towards maximal turnover rate of the whole translation cycle.

  • 170.
    Aqvist, Johan
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
    Lind, Christoffer
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
    Sund, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
    Wallin, Goran
    Bridging the gap between ribosome structure and biochemistry by mechanistic computations2012Ingår i: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 22, nr 6, s. 815-823Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The wealth of structural and biochemical data now available for protein synthesis on the ribosome presents major new challenges for computational biochemistry. Apart from technical difficulties in modeling ribosome systems, the complexity of the overall translation cycle with a multitude of different kinetic steps presents a formidable problem for computational efforts where we have only seen the beginning. However, a range of methodologies including molecular dynamics simulations, free energy calculations, molecular docking and quantum chemical approaches have already been put to work with promising results. In particular, the combined efforts of structural biology, biochemistry, kinetics and computational modeling can lead towards a quantitative structure-based description of translation.

  • 171.
    Arand, M.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. PROGRAM IN STRUCTURAL MOLECULAR BIOLOGY.
    Cronin, A
    Oesch, F
    Mowbray, Sherry L
    Department of Molecular Biosciences, Swedish University of Agricultural Sciences.
    Jones, T. Alwyn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    The telltale structures of epoxide hydrolases2003Ingår i: Drug metabolism reviews (Softcover ed.), ISSN 0360-2532, E-ISSN 1097-9883, Vol. 35, nr 4, s. 365-383Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Traditionally, epoxide hydrolases (EH) have been regarded as xenobiotic-metabolizing enzymes implicated in the detoxification of foreign compounds. They are known to play a key role in the control of potentially genotoxic epoxides that arise during metabolism of many lipophilic compounds. Although this is apparently the main function for the mammalian microsomal epoxide hydrolase (mEH), evidence is now accumulating that the mammalian soluble epoxide hydrolase (sEH), despite its proven role in xenobiotic metabolism, also has a central role in the formation and breakdown of physiological signaling molecules. In addition, a certain class of microbial epoxide hydrolases has recently been identified that is an integral part of a catabolic pathway, allowing the use of specific terpens as sole carbon sources. The recently available x-ray structures of a number of EHs mirror their respective functions: the microbial terpen EH differs in its fold from the canonical α/β hydrolase fold of the xenobiotic-metabolizing mammalian EHs. It appears that the latter fold is the perfect solution for the efficient detoxification of a large variety of structurally different epoxides by a single enzyme, whereas the smaller microbial EH, which has a particularly high turnover number with its prefered substrate, seems to be the better solution for the hydrolysis of one specific substrate. The structure of the sEH also includes an additional catalytic domain that has recently been shown to possess phosphatase activity. Although the physiological substrate for this second active site has not been identified so far, the majority of known phosphatases are involved in signaling processes, suggesting that the sEH phosphatase domain also has a role in the regulation of physiological functions.

  • 172. Arand, Michael
    et al.
    Hallberg, B Martin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Zou, Jinyu
    Bergfors, Terese
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Oesch, Franz
    van der Werf, Mariette
    de Bont, Jan A M
    Jones, T Alwyn
    Mowbray, Sherry L
    Department of Molecular Biosciences, Swedish University of Agricultural Science.
    Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site.2003Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, nr 11, s. 2583-2592Artikel i tidskrift (Refereegranskat)
  • 173.
    Ardell, David H
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Centrum för bioinformatik.
    SCANMS: adjusting for multiple comparisons in sliding window neutrality tests.2004Ingår i: Bioinformatics, ISSN 1367-4803, Vol. 20, nr 12, s. 1986-8Artikel i tidskrift (Övrigt vetenskapligt)
  • 174.
    Ardell, David H.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Centrum för bioinformatik. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution.
    Andersson, Siv G. E.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution.
    TFAM detects co-evolution of tRNA identity rules with lateral transfer of histidyl-tRNA sythetase2006Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, nr 3, s. 893-904Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We present TFAM, an automated, statistical method to classify the identity of tRNAs. TFAM, currently optimized for bacteria, classifies initiator tRNAs and predicts the charging identity of both typical and atypical tRNAs such as suppressors with high confidence. We show statistical evidence for extensive variation in tRNA identity determinants among bacterial genomes due to variation in overall tDNA base content. With TFAM we have detected the first case of eukaryotic-like tRNA identity rules in bacteria. An alpha-proteobacterial clade encompassing Rhizobiales, Caulobacter crescentus and Silicibacter pomeroyi, unlike a sister clade containing the Rickettsiales, Zymomonas mobilis and Gluconobacter oxydans, uses the eukaryotic identity element A73 instead of the highly conserved prokaryotic element C73. We confirm divergence of bacterial histidylation rules by demonstrating perfect covariation of alpha-proteobacterial tRNA(His) acceptor stems and residues in the motif IIb tRNA-binding pocket of their histidyl-tRNA synthetases (HisRS). Phylogenomic analysis supports lateral transfer of a eukaryotic-like HisRS into the alpha-proteobacteria followed by in situ adaptation of the bacterial tDNA(His) and identity rule divergence. Our results demonstrate that TFAM is an effective tool for the bioinformatics, comparative genomics and evolutionary study of tRNA identity.

  • 175.
    Ardell, David H
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Kirsebom, Leif A
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    The genomic pattern of tDNA operon expression in E. coli2005Ingår i: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 1, nr 1, s. e12-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In fast-growing microorganisms, a tRNA concentration profile enriched in major isoacceptors selects for the biased usage of cognate codons. This optimizes translational rate for the least mass invested in the translational apparatus. Such translational streamlining is thought to be growth-regulated, but its genetic basis is poorly understood. First, we found in reanalysis of the E. coli tRNA profile that the degree to which it is translationally streamlined is nearly invariant with growth rate. Then, using least squares multiple regression, we partitioned tRNA isoacceptor pools to predicted tDNA operons from the E. coli K12 genome. Co-expression of tDNAs in operons explains the tRNA profile significantly better than tDNA gene dosage alone. Also, operon expression increases significantly with proximity to the origin of replication, oriC, at all growth rates. Genome location explains about 15% of expression variation in a form, at a given growth rate, that is consistent with replication-dependent gene concentration effects. Yet the change in the tRNA profile with growth rate is less than would be expected from such effects. We estimated per-copy expression rates for all tDNA operons that were consistent with independent estimates for rDNA operons. We also found that tDNA operon location, and the location dependence of expression, were significantly different in the leading and lagging strands. The operonic organization and genomic location of tDNA operons are significant factors influencing their expression. Nonrandom patterns of location and strandedness shown by tDNA operons in E. coli suggest that their genomic architecture may be under selection to satisfy physiological demand for tRNA expression at high growth rates.

  • 176.
    Ardell, David Herman
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Centrum för bioinformatik.
    Informatic Approaches to Molecular Translation2005Ingår i: Intelligent Information Processing and Web Mining: Advances in Soft Computing, Proceedings of the IIS'2005 Symposium, 2005, s. 684-Konferensbidrag (Övrigt vetenskapligt)
  • 177.
    Argaman, L., Hershberg, R., Vogel, J., Bejerano, G., Wagner, E.G.H., Margalit, H. and Altuvia, S.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Novel small RNA-encoding genes in the intergenic regions of Escherichia coli.2001Ingår i: Curr. Biol., Vol. 11, nr 12, s. 941-950Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Small, untranslated RNA molecules were identified initially in bacteria, but examples can be found in all kingdoms of life. These RNAs carry out diverse functions, and many of them are regulators of gene expression. Genes encoding small, untra

  • 178. Arike, Liisa
    et al.
    Recktenwald, Christian V
    Liu, Jingyi
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Trillo-Muyo, Sergio
    Svärd, Staffan G
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hansson, Gunnar C
    MUC2 mucin is cleaved by Giardia intestinalis cysteine protease14019Manuskript (preprint) (Övrigt vetenskapligt)
  • 179.
    Arkov, AL
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Freistroffer, DV
    Ehrenberg, M
    Murgola, EJ
    Mutations in RNAs of both ribosomal subunits cause defects in translation termination1998Ingår i: EMBO JOURNAL, ISSN 0261-4189, Vol. 17, nr 5, s. 1507-1514Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    Mutations in RNAs of both subunits of the Escherichia coil ribosome caused defects in catalysis of peptidyl-tRNA hydrolysis in a realistic in vitro termination system. Assaying the two codon-dependent cytoplasmic proteins that drive termination, RF1 and R

  • 180.
    Arkov, AL
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Freistroffer, DV
    Pavlov, MY
    Ehrenberg, M
    Murgola, EJ
    Mutations in conserved regions of ribosomal RNAs decrease the productive association of peptide-chain release factors with the ribosome during translation termination2000Ingår i: BIOCHIMIE, ISSN 0300-9084, Vol. 82, nr 8, s. 671-682Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Early studies provided evidence that peptide-chain release factors (RFs) bind to both ribosomal subunits and trigger translation termination. Although many ribosomal proteins have been implicated in termination, very few data present direct biochemical ev

  • 181.
    Artemenko, Konstantin A.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Zubarev, Alexander R.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Samgina, Yu
    Lebedev, T
    Savitski, Mikhail M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Zubarev, Roman A.
    Two dimensional mass mapping as a general method of data representation in comprehensive analysis of complex molecular mixtures.2009Ingår i: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 81, nr 10, s. 3738-3745Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A recent proteomics-grade (95%+ sequence reliability) high-throughput de novo sequencing method utilizes the benefits of high resolution, high mass accuracy, and the use of two complementary fragmentation techniques collision-activated dissociation (CAD) and electron capture dissociation (ECD). With this high-fidelity sequencing approach, hundreds of peptides can be sequenced de novo in a single LC-MS/MS experiment. The high productivity of the new analysis technique has revealed a new bottleneck which occurs in data representation. Here we suggest a new method of data analysis and visualization that presents a comprehensive picture of the peptide content including relative abundances and grouping into families. The 2D mass mapping consists of putting the molecular masses onto a two-dimensional bubble plot, with the relative monoisotopic mass defect and isotopic shift being the axes and with the bubble area proportional to the peptide abundance. Peptides belonging to the same family form a compact group on such a plot, so that the family identity can in many cases be determined from the molecular mass alone. The performance of the method is demonstrated on the high-throughput analysis of skin secretion from three frogs, Rana ridibunda, Rana arvalis, and Rana temporaria. Two dimensional mass maps simplify the task of global comparison between the species and make obvious the similarities and differences in the peptide contents that are obscure in traditional data presentation methods. Even biological activity of the peptide can sometimes be inferred from its position on the plot. Two dimensional mass mapping is a general method applicable to any complex mixture, peptide and nonpeptide alike.

  • 182.
    Artursson, Per
    et al.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.
    Knight, Stefan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Breaking the intestinal barrier to deliver drugs2015Ingår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 347, nr 6223, s. 716-717Artikel i tidskrift (Övrigt vetenskapligt)
  • 183.
    Arwidsson, Ola
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Hughes, Diarmaid
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Evidence against reciprocal recombination as the basis for tuf gene conversion in Salmonella enterica serovar Typhimurium2004Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 338, nr 3, s. 463-467Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The duplicate tuf genes on the Salmonella enterica serovar Typhimurium chromosome co-evolve by a RecA-, RecB-dependent gene conversion mechanism. Gene conversion is defined as a non-reciprocal transfer of genetic information. However, in a replicating bacterial chromosome there is a possibility that a reciprocal genetic exchange between different tuf genes sitting on sister chromosomes could result in "apparent" gene conversion. We asked whether the major mechanism of tuf gene conversion was classical or apparent. We devised a genetic selection that allowed us to isolate and examine both expected products from a reciprocal recombination event between the tuf genes. Using this selection we tested within individual cultures for a correlation in the frequency of jackpots as expected if recombination were reciprocal. We found no correlation, either in the frequency of each type of recombinant product, or in the DNA sequences of the products resulting from each recombination event. We conclude that the evidence argues in favor of a non-reciprocal gene conversion mechanism as the basis for tuf gene co-evolution.

  • 184.
    Asp, Michaela
    et al.
    KTH Royal Inst Technol, Div Gene Technol, Sci Life Lab, Stockholm, Sweden..
    Salmen, Fredrik
    KTH Royal Inst Technol, Div Gene Technol, Sci Life Lab, Stockholm, Sweden..
    Ståhl, Patrik L.
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden..
    Vickovic, Sanja
    KTH Royal Inst Technol, Div Gene Technol, Sci Life Lab, Stockholm, Sweden..
    Felldin, Ulrika
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Löfling, Marie
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Navarro, Jose Fernandez
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden..
    Maaskola, Jonas
    KTH Royal Inst Technol, Div Gene Technol, Sci Life Lab, Stockholm, Sweden..
    Eriksson, Maria J.
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Physiol, Stockholm, Sweden..
    Persson, Bengt
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik. Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Stockholm, Sweden..
    Corbascio, Matthias
    Karolinska Univ Hosp, Dept Cardiothorac Surg & Anesthesiol, Solna, Sweden..
    Persson, Hans
    Danderyd Hosp, Dept Cardiol, Stockholm, Sweden.;Karolinska Inst, Danderyd Hosp, Dept Clin Sci, Stockholm, Sweden..
    Linde, Cecilia
    Karolinska Inst, Dept Med, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Cardiol, Stockholm, Sweden..
    Lundeberg, Joakim
    KTH Royal Inst Technol, Div Gene Technol, Sci Life Lab, Stockholm, Sweden..
    Spatial detection of fetal marker genes expressed at low level in adult human heart tissue2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 12941Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Heart failure is a major health problem linked to poor quality of life and high mortality rates. Hence, novel biomarkers, such as fetal marker genes with low expression levels, could potentially differentiate disease states in order to improve therapy. In many studies on heart failure, cardiac biopsies have been analyzed as uniform pieces of tissue with bulk techniques, but this homogenization approach can mask medically relevant phenotypes occurring only in isolated parts of the tissue. This study examines such spatial variations within and between regions of cardiac biopsies. In contrast to standard RNA sequencing, this approach provides a spatially resolved transcriptome- and tissue-wide perspective of the adult human heart, and enables detection of fetal marker genes expressed by minor subpopulations of cells within the tissue. Analysis of patients with heart failure, with preserved ejection fraction, demonstrated spatially divergent expression of fetal genes in cardiac biopsies.

  • 185. Aspegren, Anders
    et al.
    Hinas, Andrea
    Larsson, Pontus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Larsson, Anders
    Söderbom, Fredrik
    Novel non-coding RNAs in Dictyostelium discoideum and their expression during development2004Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 32, nr 15, s. 4646-4656Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The quest for non-coding RNAs (ncRNAs) in the last few years has revealed a surprisingly large number of small RNAs belonging to previously known as well as entirely novel classes. Computational and experimental approaches have uncovered new ncRNAs in all kingdoms of life. In this work, we used a shotgun cloning approach to construct full-length cDNA libraries of small RNAs from the eukaryotic model organism Dictyostelium discoideum. Interestingly, two entirely novel classes of RNAs were identified of which one is developmentally regulated. The RNAs within each class share conserved 5'- and 3'-termini that can potentially form stem structures. RNAs of both classes show predominantly cytoplasmic localization. In addition, based on conserved structure and/or sequence motifs, several of the identified ncRNAs could be divided into classes known from other organisms, e.g. 18 small nucleolar RNA candidates (17 box C/D, of which a few are developmentally regulated, and one box H/ACA). Two ncRNAs showed a high degree of similarity to the small nuclear U2 RNA and signal recognition particle RNA (SRP RNA), respectively. Furthermore, the majority of the regions upstream of the sequences encoding the isolated RNAs share conserved motifs that may constitute new promoter elements.

  • 186.
    Astvaldsson, Asgeir
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hultenby, Kjell
    Karolinska Inst, Dept Lab Med, Stockholm, Sweden.
    Svärd, Staffan G.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala Univ, BMC, Dept Cell & Mol Biol, Uppsala, Sweden.
    Jerlström-Hultqvist, Jon
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Proximity Staining Using Enzymatic Protein Tagging in Diplomonads2019Ingår i: MSPHERE, ISSN 2379-5042, Vol. 4, nr 2, artikel-id e00153-19Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis. Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) to enable the study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags require light-induced activation (1, 2) or are inactive in many cellular compartments (3), while ascorbate peroxidase has not been shown to have those limitations. Here, we have optimized the in vivo activities of two versions of pea ascorbate peroxidase (APX(W41F) and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis. We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3'-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida. Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with superresolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM, and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalis. IMPORTANCE The function of many proteins is intrinsically related to their cellular location. Novel methods for ascertainment of the ultrastructural location of proteins have been introduced in recent years, but their implementation in protists has so far not been readily realized. Here, we present an optimized proximity labeling protocol using the APEX system in the salmon pathogen Spironucleus salmonicida. This protocol was also applicable to the human pathogen Giardia intestinalis. Both organisms required extraneous addition of hemin to the growth medium to enable detectable peroxidase activity. Further, we saw no inherent limitation in labeling efficiency coupled to the cellular compartment, as evident with some other proximity labeling systems. We anticipate that the APEX proximity labeling system might offer a great resource to establish the ultrastructural localization of proteins across genetically tractable protists but might require organism-specific labeling conditions.

  • 187.
    Astvaldsson, Asgeir
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Hultenby, Kjell
    Svärd, Staffan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Jerlström-Hultqvist, Jon
    Proximity Staining using Enzymatic Protein Tagging in Diplomonads2019Ingår i: mSphere, E-ISSN 2379-5042, Vol. 4, nr 2, artikel-id e00153-19Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis. Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM), to enable study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags requires light-induced activation (1, 2) or are inactive in many cellular compartments (3) while ascorbate peroxidase has not been shown to have those limitations. Here we have optimized the in vivo activity of two versions of pea ascorbate peroxidase (APXW41F and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis. We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3’-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida. Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with super-resolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalis.

  • 188.
    Astvaldsson, Asgeir
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Stairs, Courtney W.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Xu, Feifei
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution.
    Haag, Lars
    Alfjorden, Anders
    Jansson, Eva
    Ettema, Thijs
    Svärd, Staffan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Dual transcriptomic analysis of Spironucleus salmonicida-infected salmon cells identifies putative virulence factors and host responsesManuskript (preprint) (Övrigt vetenskapligt)
  • 189.
    Atkinson, Gemma C.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik.
    Baldauf, Sandra L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik.
    Hauryliuk, Vasili
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components2008Ingår i: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 8, s. 290-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Members of the eukaryote/archaea specific eRF1 and eRF3 protein families have central roles in translation termination. They are also central to various mRNA surveillance mechanisms, together with the eRF1 paralogue Dom34p and the eRF3 paralogues Hbs1p and Ski7p. We have examined the evolution of eRF1 and eRF3 families using sequence similarity searching, multiple sequence alignment and phylogenetic analysis. Results: Extensive BLAST searches confirm that Hbs1p and eRF3 are limited to eukaryotes, while Dom34p and eRF1 (a/eRF1) are universal in eukaryotes and archaea. Ski7p appears to be restricted to a subset of Saccharomyces species. Alignments show that Dom34p does not possess the characteristic class-1 RF minidomains GGQ, NIKS and YXCXXXF, in line with recent crystallographic analysis of Dom34p. Phylogenetic trees of the protein families allow us to reconstruct the evolution of mRNA surveillance mechanisms mediated by these proteins in eukaryotes and archaea. Conclusion: We propose that the last common ancestor of eukaryotes and archaea possessed Dom34p-mediated no-go decay (NGD). This ancestral Dom34p may or may not have required a trGTPase, mostly like a/eEF1A, for its delivery to the ribosome. At an early stage in eukaryotic evolution, eEF1A was duplicated, giving rise to eRF3, which was recruited for translation termination, interacting with eRF1. eRF3 evolved nonsense-mediated decay (NMD) activity either before or after it was again duplicated, giving rise to Hbs1p, which we propose was recruited to assist eDom34p in eukaryotic NGD. Finally, a third duplication within ascomycete yeast gave rise to Ski7p, which may have become specialised for a subset of existing Hbs1p functions in non-stop decay (NSD). We suggest Ski7p-mediated NSD may be a specialised mechanism for counteracting the effects of increased stop codon read-through caused by prion-domain [ PSI+] mediated eRF3 precipitation.

  • 190.
    Attwood, T.K.
    et al.
    Faculty of Life Sciences & School of Computer Science, University of Manchester.
    Gisel, A
    Institute for Biomedical Technologies, CNR, Italy.
    Eriksson, Nils-Einar
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Bongcam-Rudloff, Erik
    Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences.
    Concepts, Historical Milestones and the Central Place of Bioinformatics in Modern Biology: A European Perspective2011Ingår i: Bioinformatics: Trends and Methodologies / [ed] Mahmood A. Mahdavi, InTech, 2011, s. 3-26Kapitel i bok, del av antologi (Refereegranskat)
  • 191.
    Ausmees, Nora
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Intermediate filament-like cytoskeleton of Caulobacter crescentus2006Ingår i: Journal of Molecular Microbiology and Biotechnology, ISSN 1464-1801, Vol. 11, nr 3-5, s. 152-158Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Eukaryotic cytoskeleton consists of three main types of filaments: actin microfilaments, microtubules and intermediate filaments (IFs). Actin and tubulin-like proteins are also found in bacteria where they perform diverse cytoskeletal functions. IFs, however, are considered to be a characteristic constituent of metazoan cells only, where they (among other functions) are involved in determination and maintenance of cell shape and cellular integrity. Surprisingly, a coiled coil-rich protein called crescentin was recently shown to play a key role in determining the complex curved and helical cell shapes of the bacterium Caulobacter crescentus, and to exhibit several characteristic properties of animal IF proteins. First, the arrangement of the coiled coil domains of crescentin closely resembles the tripartite molecular architecture of IF proteins. Second, crescentin also possesses the defining biochemical property of IF proteins to assemble into 10-nm-wide filaments in vitro without cofactors. Furthermore, crescentin forms a higher-order helical structure in vivo, which is localized asymmetrically along the concave side of the cell. In close association with the cell membrane, the crescentin structure promotes the helical growth of the cell and thereby determines a curved or a helical shape, depending on the length of the cell. The unexpected finding of an IF-like element in a bacterium raises several interesting questions concerning, for example, the molecular mechanisms whereby complex and asymmetric cell shapes are generated by different bacteria, or the functional and evolutionary relatedness of crescentin to animal IF proteins.

  • 192.
    Ausmees, Nora
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wahlstedt, Helene
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Bagchi, Sonchita
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Elliot, Marie A.
    Buttner, Mark J.
    Flärdh, Klas
    SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa2007Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 65, nr 6, s. 1458-1473Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK-family protein) is characterized. Deletion of smeA-sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z-ring assembly. The presence of smeA-like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK- or Tra-like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation.

  • 193.
    Aveskogh, Maria
    et al.
    Department of medical immunology and microbiology. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Lützelschwab, Claudia
    Department of medical immunology and microbiology.
    Huang, Mallen R.
    Department of medical immunology and microbiology.
    Hellman, Lars
    Department of medical immunology and microbiology.
    Characterization of cDNA clones encoding mouse proteinase 3 (myeloblastine) and cathepsin G1997Ingår i: Immunogenetics, ISSN 0093-7711, E-ISSN 1432-1211, Vol. 46, nr 3, s. 181-191Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Serine proteases are the most abundant granule constituents of several major hematopoietic cell lineages. Due to their high abundance and their strict tissue specificity they have become important phenotypic cell markers used for studies of various aspects of hematopietic cell development. Using a polymerase chain reaction (PCR)-based strategy for the isolation of trypsin-related serine proteases, we were able to isolate cDNAs for two of the major neutrophil and monocyte serine proteases in the mouse, cathepsin G and mouse protease 3 (myeloblastin). The internal PCR fragments were used as probes to screen a mouse mast cell cDNA library and a cDNA library originating from a mouse monocytic cell line (WEHI-274.1). Full-length cDNAs for mouse cathepsin G and proteinase 3 were isolated and their complete sequences were determined. Northern blot analysis revealed expression of cathepsin G in immature cells of the monocyte macrophage lineage but also in the connective tissue mast cell line MTC. Proteinase 3 was expressed in several cell lines of myelo-monocytic origin and in one B-cell line, but not in any of the other cell lines tested. The isolation of cDNAs for mouse cathepsin G and mouse proteinase 3, together with the previous characterization of the gene for mouse N-elastase, and the entire or partial amino acid sequences for porcine azurocidine, equine N-elastase and proteinase 3, rat, dog, and rabbit cathepsin Gs in evolutionary relatively distantly related mammalian species, indicates that these four members of the serine protease family have been maintained for more than 100 million years of mammalian evolution. This latter finding indicates a strong evolutionary pressure to maintain specific immune functions associated with these neutrophil and monocyte proteases. All amino acid positions of major importance for the cleavage site selection have also been fully conserved between mouse and human proteinase 3 and a few minor changes have occurred between mouse and human cathepsin G.

  • 194.
    Aveskogh, Maria
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pilström, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Cloning and structural analysis of IgM (μ chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica1999Ingår i: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 23, nr 7-8, s. 597-606Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (γ chain), IgE (ε chain) and IgA (α chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (μ chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.

  • 195. Avesson, Lotta
    et al.
    Reimegård, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, Gerhart Eduard Heinrich
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Söderbom, Fredrik
    Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs2012Ingår i: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 18, nr 10, s. 1771-1782Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a common origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. One example is miRNAs. Their regulation of protein coding genes has been shown to play a vital role in plants and animals but little is known about their role in other organisms. The single cell social amoeba Dictyostelium discoideum could hold the answers to some questions regarding the evolution and function of small RNA pathways. Here we report deep sequencing of small RNAs from three developmental stages of D. discoideum. Analyses of these libraries as well as experimental data reveal the expression of a number of miRNAs, several which have distinct expression patterns during development. We also find miRNAs processed from a hairpin originating from a repetitive element that we believe could represent a pathway for the generation of new miRNAs.

  • 196. Azuaje, Jhonny
    et al.
    Carbajales, Carlos
    Gonzalez-Gomez, Manuel
    Coelho, Alberto
    Caamano, Olga
    Gutierrez-de-Teran, Hugo
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
    Sotelo, Eddy
    Pyrazin-2(1H)-ones as a novel class of selective A3 adenosine receptor antagonists2015Ingår i: Future Medicinal Chemistry, ISSN 1756-8919, E-ISSN 1756-8927, Vol. 7, nr 11, s. 1373-1380Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: A(3)AR antagonists are promising drug candidates as neuroprotective agents as well as for the treatment of inflammation or glaucoma. The most widely known A(3)AR antagonists are derived from polyheteroaromatic scaffolds, which usually show poor pharmacokinetic properties. Accordingly, the identification of structurally simple A(3)AR antagonists by the exploration of novel diversity spaces is a challenging goal. Results: A convergent and efficient Ugi-based multicomponent approach enabled the discovery of pyrazin-2(1H)-ones as a novel class of A(3)AR antagonists. A combined experimental/computational strategy accelerated the establishment of the most salient features of the structure-activity and structure-selectivity relationships in this series. Conclusion: The optimization process provided pyrazin-2(1H)-ones with improved affinity and a plausible hypothesis regarding their binding modes was proposed.

  • 197.
    Azuaje, Jhonny
    et al.
    Univ Santiago de Compostela, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain.;Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Jespers, Willem
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Yaziji, Vicente
    Univ Santiago de Compostela, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain.;Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Mallo, Ana
    Univ Santiago de Compostela, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain.;Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Majellaro, Maria
    Univ Santiago de Compostela, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain.;Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Caamano, Olga
    Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Loza, Maria I.
    Univ Santiago de Compostela, Ctr Singular Invest Med Mol & Enfermedades Cronic, Santiago De Compostela 15782, Spain..
    Cadavid, Maria I.
    Univ Santiago de Compostela, Ctr Singular Invest Med Mol & Enfermedades Cronic, Santiago De Compostela 15782, Spain..
    Brea, Jose
    Univ Santiago de Compostela, Ctr Singular Invest Med Mol & Enfermedades Cronic, Santiago De Compostela 15782, Spain..
    Åqvist, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Sotelo, Eddy
    Univ Santiago de Compostela, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain.;Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Gutiérrez-de-Terán, Hugo
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Effect of Nitrogen Atom Substitution in A(3) Adenosine Receptor Binding: N-(4,6-Diarylpyridin-2-yl)acetamides as Potent and Selective Antagonists2017Ingår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 60, nr 17, s. 7502-7511Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We report the first family of 2-acetamidopyridines as potent and selective A, adenosine receptor (AR) antagonists. The computer -assisted design was focused on the bioisosteric replacement of the N1 atom by a CH group in a previous series of diarylpyrimidines. Some of the generated 2-acetamidopyridines elicit an antagonistic effect with excellent affinity (K-j < 10 nM) and outstanding selectivity profiles, providing an alternative and simpler chemical scaffold to the parent series of diarylpyrimidines. In addition, using molecular dynamics and free energy perturbation simulations, we elucidate the effect of the second nitrogen of the parent diarylpyrimidines, which is revealed as a stabilizer of a water network in the binding site. The discovery of 2,6-diaryl-2-acetamidopyridines represents a step forward in the search of chemically simple, potent, and selective antagonists for the hA(3)AR, and exemplifies the benefits of a joint theoretical- experimental approach to identify novel hA(3)AR antagonists through succinct and efficient synthetic methodologies.

  • 198.
    Bagchi, Sonchita
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Coiled coil Cytoskeleton in Bacterial Cell Architecture: Studies of Growth and Development in Streptomyces2011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Bacterial cytoskeleton is an exciting and relatively new field of research. Recent findings have proven that microbes are well-organized and neatly structured organisms. In this study we have shown that intermediate filament-like proteins with a characteristic rod domain architecture of coiled coil segments separated by non-coiled coil linkers, are widely spread among bacteria.

    We identified and characterized an intermediate filament-like protein (named FilP after filamentous protein) in Streptomyces coelicolor. It shares the characteristic biochemical property of eukaryotic intermediate filaments of formation of spontaneous filaments in vitro without requiring any energy or co-factor. We have provided here a preliminary model of its assembly in vitro. FilP also forms in vivo filaments in S. coelicolor hyphae, which are strongest at the sub-apical location of growing vegetative hyphae. We have proposed that FilP cytoskeletal network provides rigidity to the hyphae, especially at the growing tips, by interacting with an essential coiled coil protein DivIVA and possibly other partner elements, yet to be found.

    S. coelicolor is a well-studied model organism with a complicated life cycle. It germinates from a spore and spreads by forming branched vegetative hyphae. Lack of nutrients in the environment initiates formation of aerial hyphae in the air, perpendicular to the vegetative ones. The aerial hyphae differentiate into spore chains and eventually grey-pigmented dispersed individual spores are released. The signals involved in sporulation including cell division and chromosome segregation are not clear yet. We characterized here a novel locus consisting of two genes: a small putative membrane protein with no defined function, named SmeA and a member of the SpoIIIE/FtsK family, called SffA. The expression of this locus appears to be dependent on whiA and whiG-whiH-whiI pathways. This finding is intriguing as it can provide insight to the relationship between two apparently unrelated pathways, both leading to the same function of septation and maturation during sporulation.

    Delarbeten
    1. Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces
    Öppna denna publikation i ny flik eller fönster >>Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces
    2008 (Engelska)Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, nr 4, s. 1037-1050Artikel i tidskrift (Refereegranskat) Published
    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.

    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-107121 (URN)10.1111/j.1365-2958.2008.06473.x (DOI)000260194300022 ()
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