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  • 1.
    Andersson, Magnus
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Svantesson, Mats
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Björnham, Oscar
    Swedish Defence Research Agency (FOI), SE-906 21 Umeå, Sweden.
    Badahdah, Arwa
    Department of Oral Biology, Boston University School of Dental Medicine.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bullitt, Esther
    Department of Physiology and Biophysics, Boston University School of Medicine.
    A structural basis for sustained bacterial adhesion: Biomechanical properties of CFA/I Pili2012In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 415, no 5, p. 918-928Article in journal (Refereed)
    Abstract [en]

    Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized at single organelle level the intrinsic biomechanical properties and kinetics of individual CFA/I pili, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P-pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix, and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.

  • 2.
    Antonyuk, Svetlana V
    et al.
    Molecular Biophysics Group, School of Biological Sciences, University of Liverpool.
    Strange, Richard W
    Molecular Biophysics Group, School of Biological Sciences, University of Liverpool.
    Marklund, Stefan L
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Hasnain, S Samar
    Molecular Biophysics Group, School of Biological Sciences, University of Liverpool.
    The structure of human extracellular copper-zinc superoxide dismutase at 1.7 A resolution: insights into heparin and collagen binding2009In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 388, no 2, p. 310-326Article in journal (Refereed)
    Abstract [en]

    Extracellular superoxide dismutase (SOD3) is a homotetrameric copper- and zinc-containing glycoprotein with affinity for heparin. The level of SOD3 is particularly high in blood vessel walls and in the lungs. The enzyme has multiple roles including protection of the lungs against hyperoxia and preservation of nitric oxide. The common mutation R213G, which reduces the heparin affinity of SOD3, is associated with increased risk of myocardial infarctions and stroke. We report the first crystal structure of human SOD3 at 1.7 A resolution. The overall subunit fold and the subunit-subunit interface of the SOD3 dimer are similar to the corresponding structures in Cu-Zn SOD (SOD1). The metal-binding sites are similar to those found in SOD1, but with Asn180 replacing Thr137 at the Cu-binding site and a much shorter loop at the zinc-binding site. The dimers form a functional homotetramer that is fashioned through contacts between two extended loops on each subunit. The N- and C-terminal end regions required for tetramerisation and heparin binding, respectively, are highly flexible. Two grooves fashioned by the tetramer interface are suggestive as the probable sites for heparin and collagen binding.

  • 3. Blanch, Ewan W
    et al.
    Morozova-Roche, Ludmilla A
    Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford.
    Cochran, Duncan A
    Doig, Andrew J
    Hecht, Lutz
    Barron, Laurence D
    Is polyproline II helix the killer conformation? A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme.2000In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 301, no 2, p. 553-563Article in journal (Refereed)
    Abstract [en]

    The amyloidogenic prefibrillar partially denatured intermediate of human lysozyme, prepared by heating the native protein to 57 degrees C at pH 2.0, was studied using Raman optical activity (ROA). A positive band in the room temperature ROA spectrum of the native protein at approximately 1345 cm(-1), assigned to a hydrated form of alpha-helix, is not present in that of the prefibrillar intermediate, where a new strong positive band at approximately 1318 cm(-1) appears instead that is assigned to the poly(l-proline) II (PPII)-helical conformation. A sharp negative band at approximately 1241 cm(-1) in the native protein, assigned to beta-strand, shows little change in the ROA spectrum of the prefibrillar intermediate. The disappearance of a positive ROA band at approximately 1551 cm(-1) assigned to vibrations of tryptophan side-chains indicates that major conformational changes have occurred among the five tryptophan residues present in human lysozyme, four of which are located in the alpha-domain. The various ROA data suggest that a substantial loss of tertiary structure has occurred in the prefibrillar intermediate and that this is located more in the alpha-domain than in the beta-domain. There is no evidence for any increase in beta-structure. The ROA spectrum of hen lysozyme, which does not form amyloid fibrils so readily, remains much more native-like on heating to 57 degrees C at pH 2.0. The thermal behaviour of the alanine-rich alpha-helical peptide AK21 in aqueous solution was found to be similar to that of human lysozyme. Hydrated alpha-helix therefore appears to readily undergo a conformational change to PPII structure on heating, which may be a key step in the conversion of alpha-helix into beta-sheet in the formation of amyloid fibrils in human lysozyme. Since it is extended, flexible, lacks intrachain hydrogen bonds and is fully hydrated in aqueous solution, PPII helix has the appropriate characteristics to be implicated as a critical conformational element in many conformational diseases. Disorder of the PPII type may be a sine qua non for the formation of regular fibrils; whereas the more dynamic disorder of the random coil may lead only to amorphous aggregates.

  • 4.
    Bokvist, Marcus
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lindström, Fredrick
    Umeå University, Faculty of Science and Technology, Chemistry.
    Watts, Anthony
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Two Types of Alzheimer’s β-Amyloid (1–40) Peptide Membrane Interactions: Aggregation Preventing Transmembrane Anchoring Versus Accelerated Surface Fibril Formation2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 335, no 4, p. 1039-1049Article in journal (Refereed)
    Abstract [en]

    The 39–42 amino acid long, amphipathic amyloid-β peptide (Aβ) is one of the key components involved in Alzheimer's disease (AD). In the neuropathology of AD, Aβ presumably exerts its neurotoxic action via interactions with neuronal membranes. In our studies a combination of 31P MAS NMR (magic angle spinning nuclear magnetic resonance) and CD (circular dichroism) spectroscopy suggest fundamental differences in the functional organization of supramolecular Aβ1–40 membrane assemblies for two different scenarios with potential implication in AD: Aβ peptide can either be firmly anchored in a membrane upon proteolytic cleavage, thereby being prevented against release and aggregation, or it can have fundamentally adverse effects when bound to membrane surfaces by undergoing accelerated aggregation, causing neuronal apoptotic cell death. Acidic lipids can prevent release of membrane inserted Aβ1–40 by stabilizing its hydrophobic transmembrane C-terminal part (residue 29–40) in an α-helical conformation via an electrostatic anchor between its basic Lys28 residue and the negatively charged membrane interface. However, if Aβ1–40 is released as a soluble monomer, charged membranes act as two-dimensional aggregation-templates where an increasing amount of charged lipids (possible pathological degradation products) causes a dramatic accumulation of surface-associated Aβ1–40 peptide followed by accelerated aggregation into toxic structures. These results suggest that two different molecular mechanisms of peptide–membrane assemblies are involved in Aβ′s pathophysiology with the finely balanced type of Aβ–lipid interactions against release of Aβ from neuronal membranes being overcompensated by an Aβ–membrane assembly which causes toxic β-structured aggregates in AD. Therefore, pathological interactions of Aβ peptide with neuronal membranes might not only depend on the oligomerization state of the peptide, but also the type and nature of the supramolecular Aβ–membrane assemblies inherited from Aβ′s origin.

  • 5.
    Bosco, Daryl A
    et al.
    Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA.
    Eisenmesser, Elan Zohar
    Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA.
    Clarkson, Michael W
    Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Labeikovsky, Wladimir
    Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA.
    Millet, Oscar
    Protein Engineering Network Center of Excellence and Departments of Medical Genetics, Biochemistry, Canada.
    Kern, Dorothee
    Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA.
    Dissecting the microscopic steps of the cyclophilin a enzymatic cycle on the biological substrate HIV-capsid by NMR2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 403, no 5, p. 723-38Article in journal (Refereed)
    Abstract [en]

    Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active site residues not only reduces the catalytic activity of these enzymes, but also dramatically affects substrate binding. Employing the cyclophilin A (CypA) PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the HIV-1 capsid (CA(N)) protein, we demonstrate here how to dissect residue specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of CypA active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based on a peptide assay only, catalyze the HIV capsid with wild-type activity, but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.

  • 6.
    Brorsson, Ann-Christin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kjellson, Annika
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Aronsson, Göran
    Biopool, Umeå, Sweden.
    Sethson, Ingmar
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hambraeus, Charlotta
    University of Southern Stockholm, Center for Structural Biochemistry, Huddinge, Sweden.
    Jonsson, Bengt-Harald
    Molecular Biotechnology/IFM, Linköping University, Linköping, Sweden.
    The “Two-State folder” MerP forms partially unfolded structures that show temperature dependent hydrogen exchange2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 340, no 2, p. 333-344Article in journal (Refereed)
    Abstract [en]

    We have analysed the folding energy landscape of the 72 amino acid protein MerP by monitoring native state hydrogen exchange as a function of temperature in the range of 7-55 degrees C. The temperature dependence of the hydrogen exchange has allowed us to determine DeltaG, DeltaH and DeltaC(p) values for the conformational processes that permit hydrogen exchange. When studied with the traditional probes, fluorescence and CD, MerP appears to behave as a typical two-state protein, but the results from the hydrogen exchange analysis reveal a much more complex energy landscape. Analysis at the individual amino acid level show that exchange is allowed from an ensemble of partially unfolded structures (i.e. intermediates) in which the stabilities at the amino acid level form a broad distribution throughout the protein. The formation of partially unfolded structures might contribute to the unusually slow folding of MerP.

  • 7.
    Brorsson, Ann-Christin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lundqvist, Martin
    Sethson, Ingmar
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jonsson, Bengt-Harald
    GuHCl and NaCl-dependent hydrogen exchange in MerP reveals a well-defined core with an unusual exchange pattern2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 357, no 5, p. 1634-46Article in journal (Refereed)
    Abstract [en]

    We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (DeltaG(HX)) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions.

  • 8.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Islam, Tohidul
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gharibyan, Anna L.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Iakovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Lina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lee, Cheng Choo
    Sandblad, Linda
    Pamrén, Annelie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The Properties of Amyloid-β Fibrils Are Determined by their Path of Formation2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 13, p. 1940-1949Article in journal (Refereed)
    Abstract [en]

    Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1-40 and Aβ1-42 are the dominant forms. The fibril architectures of Aβ1-40 and Aβ1-42 differ and Aβ1-42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1-42 can be cross-templated and incorporated into the ends of Aβ1-40 fibrils, while incorporation of Aβ1-40 monomers into Aβ1-42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1-40 to incorporate into the ends of Aβ1-42 fibrils and the capacity of Aβ1-42 monomers to adopt the properties of Aβ1-40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1-42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1-40 from adopting the fibrillar properties of Aβ1-42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation.

  • 9. Bunner, Anne E
    et al.
    Nord, Stefan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wikström, P Mikael
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Williamson, James R
    The effect of ribosome assembly cofactors on in vitro 30S subunit reconstitution2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 398, no 1, p. 1-7Article in journal (Refereed)
    Abstract [en]

    Ribosome biogenesis is facilitated by a growing list of assembly cofactors, including helicases, GTPases, chaperones, and other proteins, but the specific functions of many of these assembly cofactors are still unclear. The effect of three assembly cofactors on 30S ribosome assembly was determined in vitro using a previously developed mass-spectrometry-based method that monitors the rRNA binding kinetics of ribosomal proteins. The essential GTPase Era caused several late-binding proteins to bind rRNA faster when included in a 30S reconstitution. RimP enabled faster binding of S9 and S19 and inhibited the binding of S12 and S13, perhaps by blocking those proteins' binding sites. RimM caused proteins S5 and S12 to bind dramatically faster. These quantitative kinetic data provide important clues about the roles of these assembly cofactors in the mechanism of 30S biogenesis.

  • 10.
    Byström, Anders S
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology. Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    von Gabain, A
    Björk, Glenn R
    Umeå University, Faculty of Medicine, Molecular Biology. Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species.1989In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 208, no 4, p. 575-586Article in journal (Refereed)
    Abstract [en]

    The trmD operon is a four-cistron operon in which the first and fourth genes encode ribosomal proteins S16 (rpsP) and L19 (rplS), respectively. The second gene encodes a 21,000 Mr polypeptide of unknown function and the third gene (trmD) encodes the enzyme tRNA(m1G37)methyltransferase, which catalyzes the formation of 1-methylguanosine (m1G) next to the 3' end of the anticodon (position 37) of some tRNAs in Escherichia coli. Here we show under all regulatory conditions studied, transcription initiates at one unique site, and the entire operon is transcribed into one polycistronic mRNA. Between the promoter and the first gene, rpsP, an attenuator-like structure is found (delta G = -18 kcal; 1 cal = 4.184 J), followed by four uridine residues. This structure is functional in vitro, and terminates more than two-thirds of the transcripts. The different parts of the trmD operon mRNA decay at a uniform rate. The stability of the trmD mRNA is not reduced with decreasing growth rate, which is in contrast to what has been found for other ribosomal protein mRNAs. Furthermore, earlier experiments have shown the existence of differential expression as well as non-co-ordinate regulation within the operon. Our results are consistent with the regulation of the trmD operon being due to some mechanism(s) operating at the post-transcriptional level, and do not involve differential degradation of different mRNA segments, internal promoters or internal terminators.

  • 11.
    Chen, Peng
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Qian, Qiang
    Umeå University, Faculty of Medicine, Molecular Biology.
    Zhao, Shaoping
    Isaksson, Leif A.
    Björk, Glenn R.
    Umeå University, Faculty of Medicine, Molecular Biology.
    A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine2002In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 317, no 4, p. 481-492Article in journal (Refereed)
    Abstract [en]

    Out of more than 500 sequenced cytosolic tRNAs, there is only one with an unmodified adenosine in the wobble position (position 34). The reason for this rare occurrence of A34 is that it is mostly deaminated to inosine-34 (I34). I34 is a common constituent in the wobble position of tRNAs and has a decoding capacity different from that of A34. We have isolated a mutant (proL207) of Salmonella typhimurium, in which the wobble nucleoside G34 has been replaced by an unmodified A in tRNA(Pro)(GGG), which is the only tRNA that normally reads the CCC codon. Thus, this mutant apparently has no tRNA that is considered cognate for the codon CCC. Despite this, the mutant grows normally. As expected, Pro-tRNA selection at the CCC codon in the A-site in a mutant deleted for the proL gene, which encodes the tRNA(Pro)(GGG), was severely reduced. However, in comparison this rate of selection was only slightly reduced in the proL207 mutant with its A34 containing tRNA(Pro)(AGG) suggesting that this tRNA reads CCC. Moreover, measurements of the interference by a tRNA residing in the P-site on the apparent termination efficiency at the A-site indicated that indeed the A34 containing tRNA reads the CCC codon. We conclude that A34 in a cytosolic tRNA is not detrimental to the cell and that the mutant tRNA(Pro)(AGG) is able to read the CCC codon like its wild-type counterpart tRNA(Pro)(GGG). We suggest that the decoding of the CCC codon by a 5'-AGG-3' anticodon occurs by a wobble base-pair between a protonated A34 and a C in the mRNA. Copyright 2002 Elsevier Science Ltd.

  • 12.
    Forsgren, Nina
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Lamont, Richard J
    Persson, Karina
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Two intramolecular isopeptide bonds are identified in the crystal structure of the Streptococcus gordonii SspB c-terminal domain2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, no 3, p. 740-751Article in journal (Refereed)
    Abstract [en]

    Streptococcus gordonii is a primary colonizer and is involved in the formation of dental plaque. This bacterium expresses several surface proteins. One of them is the adhesin SspB, which is a member of the Antigen I/II family of proteins. SspB is a large multi-domain protein that has interactions with surface molecules on other bacteria and on host cells, and is thus a key factor in the formation of biofilms. Here, we report the crystal structure of a truncated form of the SspB C-terminal domain, solved by single-wavelength anomalous dispersion to 1.5Å resolution. The structure represents the first of a C-terminal domain from a streptococcal Antigen I/II protein and is comprised of two structurally related β-sandwich domains, C2 and C3, both with a Ca2+ bound in equivalent positions. In each of the domains, a covalent isopeptide bond is observed between a lysine and an asparagine, a feature that is believed to be a common stabilization mechanism in Gram-positive surface proteins. S. gordonii biofilms contain attachment sites for the periodontal pathogen Porphyromonas gingivalis and the SspB C-terminal domain has been shown to have one such recognition motif, the SspB adherence region. The motif protrudes from the protein, and serves as a handle for attachment. The structure suggests several additional putative binding surfaces, and other binding clefts may be created when the fulllength protein is folded.

  • 13.
    Gharibyan, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zamotin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yanamandra, Kiran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Moskaleva, Olesya S
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Margulis, BA
    Kostanyan, IA
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 365, no 5, p. 1337-1349Article in journal (Refereed)
    Abstract [en]

    Among the newly discovered amyloid properties, its cytotoxicity plays a key role. Lysozyme is a ubiquitous protein involved in systemic amyloidoses in vivo and forming amyloid under destabilising conditions in vitro. We characterized both oligomers and fibrils of hen lysozyme by atomic force microscopy and demonstrated their dose (5–50 μM) and time-dependent (6–48 h) effect on neuroblastoma SH-SY5Y cell viability. We revealed that fibrils induce a decrease of cell viability after 6 h due to membrane damage shown by inhibition of WST-1 reduction, early lactate dehydrogenase release, and propidium iodide intake; by contrast, oligomers activate caspases after 6 h but cause the cell viability to decline only after 48 h, as shown by fluorescent-labelled annexin V binding to externalized phosphatidylserine, propidium iodide DNA staining, lactate dehydrogenase release, and by typical apoptotic shrinking of cells. We conclude that oligomers induce apoptosis-like cell death, while the fibrils lead to necrosis-like death. As polymorphism is a common property of an amyloid, we demonstrated that it is not a single uniform species but rather a continuum of cross-β-sheet-containing amyloids that are cytotoxic. An abundance of lysozyme highlights a universal feature of this phenomenon, indicating that amyloid toxicity should be assessed in all clinical applications involving proteinaceous materials.

  • 14.
    Gupta, Arun A.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Reinartz, Ines
    Karunanithy, Gogulan
    Spilotros, Alessandro
    Jonna, Venkateswara Rao
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Svergun, Dmitri I.
    Baldwin, Andrew J.
    Schug, Alexander
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 18, Part B, p. 3157-3169Article in journal (Refereed)
    Abstract [en]

    Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopHCBD) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH2. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopHCBD state has an elevated affinity for SycH2 and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH2 passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation.

  • 15. Hammer, Neal D
    et al.
    McGuffie, Bryan A
    Zhou, Yizhou
    Badtke, Matthew P
    Reineke, Ashley A
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gestwicki, Jason E
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chapman, Matthew R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The C-terminal repeating units of CsgB direct bacterial functional amyloid nucleation2012In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 422, no 3, p. 376-389Article in journal (Refereed)
    Abstract [en]

    Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β–strand-loop-β–strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB, and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the 1st, 2nd or 3rd repeating units were able to convert CsgA into fibers. However, mutants missing either the 4th or 5th repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane, but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2 and 4 self assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units.

  • 16.
    Holmner, Åsa
    et al.
    Department of Chemistry and Bioscience, Chalmers University of Technology, PO box 462, SE-40530 Göteborg, Sweden; Department of Chemistry, University of Oslo, PO box 1033 Blindern, NO-0315 Oslo, Norway.
    Askarieh, Glareh
    Ökvist, Mats
    Krengel, Ute
    Blood group antigen recognition by Escherichia coli heat-labile enterotoxin2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 371, no 3, p. 754-764Article in journal (Refereed)
    Abstract [en]

    In a number of bacterial infections, such as Helicobacter pylori, Campylobacter jejuni and Vibrio cholerae infections, a correlation between the severity of disease and blood group phenotype of infected individuals has been observed. In the present investigation, we have studied the molecular basis of this effect for enterotoxigenic Escherichia coli (ETEC) infections. ETEC are non-invasive bacteria, which act through second messenger pathways to cause diarrhea. It has been suggested that the major virulence factor of ETEC from human isolates, i.e. the human heat-labile enterotoxin (hLT), recognizes certain blood group epitopes, although the molecular basis of blood group antigen recognition is unknown. The 2.5 angstrom crystal structure of the receptor-binding B-subunit of hLT in complex with the blood group A antigen analog GalNAc alpha 3(Fuc alpha-2)Gal beta 4(Fuc alpha-3)Glc beta provides evidence of a previously unknown binding site in the native toxin. The structure reveals the molecular interactions underlying blood group antigen recognition and suggests how this protein can discriminate between different blood group epitopes. These results support the previously debated role of hLT in the blood group dependence of ETEC infections. Similar observations regarding the closely related cholera toxin in V. cholera infections are also discussed.

  • 17.
    Hägglöf, Peter
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bergström, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 335, no 3, p. 823-832Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1′ residues acting as a “bait” for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into β-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor–donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in β-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into β-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.

  • 18.
    Jia, Xueen
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gharibyan, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Anders
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Liu, Yonggang
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Neuroprotective and nootropic drug noopept rescues α-synuclein amyloid cytotoxicity2011In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 414, no 5, p. 699-712Article in journal (Refereed)
    Abstract [en]

    Parkinson's disease is a common neurodegenerative disorder characterized by α-synuclein (α-Syn)-containing Lewy body formation and selective loss of dopaminergic neurons in the substantia nigra. We have demonstrated the modulating effect of noopept, a novel proline-containing dipeptide drug with nootropic and neuroprotective properties, on α-Syn oligomerization and fibrillation by using thioflavin T fluorescence, far-UV CD, and atomic force microscopy techniques. Noopept does not bind to a sterically specific site in the α-Syn molecule as revealed by heteronuclear two-dimensional NMR analysis, but due to hydrophobic interactions with toxic amyloid oligomers, it prompts their rapid sequestration into larger fibrillar amyloid aggregates. Consequently, this process rescues the cytotoxic effect of amyloid oligomers on neuroblastoma SH-SY5Y cells as demonstrated by using cell viability assays and fluorescent staining of apoptotic and necrotic cells and by assessing the level of intracellular oxidative stress. The mitigating effect of noopept against amyloid oligomeric cytotoxicity may offer additional benefits to the already well-established therapeutic functions of this new pharmaceutical.

  • 19. Lasswitz, Lisa
    et al.
    Chandra, Naresh
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany.
    Glycomics and Proteomics Approaches to Investigate Early Adenovirus-Host Cell Interactions2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 13, p. 1863-1882Article in journal (Refereed)
    Abstract [en]

    Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.

  • 20.
    Lindberg, Magnus O
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Tångrot, Jeanette
    Umeå University, Faculty of Science and Technology, Department of Computing Science.
    Otzen, Daniel E
    Dolgikh, Dmitry A
    Finkelstein, Alexei V
    Oliveberg, Mikael
    Umeå University, Faculty of Science and Technology, Chemistry.
    Folding of circular permutants with decreased contact order: general trend balanced by protein stability2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 314, no 4, p. 891-900Article in journal (Refereed)
    Abstract [en]

    To examine the influence of contact order and stability on the refolding rate constant for two-state proteins, we have analysed the folding kinetics of the small β-α-β protein S6 and two of its circular permutants with relative contact orders of 0.19, 0.15 and 0.12. Data reveal a small but significant increase of the refolding rate constant (log kf) with decreasing contact order. At the same time, the decreased contact order is correlated to losses in global stability and alterations of the folding nucleus. When the differences in stability are accounted for by addition of Na2SO4 or by comparison of the folding kinetics at the transition mid-point, the dependence between log kf and contact order becomes stronger and follows the general correlation for two-state proteins. The observation emphasizes the combined action of topology and stability in controlling the rate constant of protein folding.

  • 21.
    Malisauskas, Mantas
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yanamandra, Kiran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lability landscape and protease resistance of human insulin amyloid: a new insight into its molecular properties2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 396, no 1, p. 60-74Article in journal (Refereed)
    Abstract [en]

    Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as AFM, thioflavin-T fluorescence, circular dichroism and gas phase electrophoretic mobility macromolecule analysis we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pHs and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding the inflection point of the growth phase; (ii) an abrupt cut-off between growth and disaggregation at pH 8-10; (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time AFM and thioflavin-T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca. 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.

  • 22.
    Malisauskas, Mantas
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zamotin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jass, Jana
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Noppe, Wim
    Dobson, Christopher M
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration2003In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 330, no 4, p. 879-890Article in journal (Refereed)
    Abstract [en]

    The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 °C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca2+ the protofilaments are present as annular structures with a diameter of 40–50 nm. In the presence of 10 mM CaCl2 the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 °C and 57 °C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70–80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1–80 and 54–125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as -synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.

  • 23.
    Morozova-Roche, Ludmilla A
    et al.
    University of Oxford, Centre for Molecular Sciences, New Chemistry Laboratory.
    Arico-Muendel, C C
    Haynie, D T
    Emelyanenko, V I
    Van Dael, H
    Dobson, C M
    Structural characterisation and comparison of the native and A-states of equine lysozyme1997In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 268, no 5, p. 903-921Article in journal (Refereed)
    Abstract [en]

    Native state 1H NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state. Native holo equine lysozyme hydrogen exchange protection factors are as large as 10(6), the most protected residues being located in elements of secondary structure. High exchange protection in the domain interface correlates with the binding of Ca2+ in this region. Equine lysozyme differs from most non-Ca2+ binding lysozymes in forming a highly populated partially folded state at low pH. The protein in this A-state at pH 2.0 has been found to bind 1-anilino-naphthalene-8-sulphonate with the enhancement of fluorescent intensity and blue shift in the spectral maximum characteristic of molten globules. NMR spectra indicate that the A-state is globally much less ordered than native equine lysozyme but does not contain significant regions of random coil structure. The amides most protected against hydrogen exchange in the A-state (protection factors up to 10(2) at 5 degrees C) correspond to residues of three of the four alpha-helices of the native state; the side-chains of these residues form a hydrophobic cluster that includes five aromatic residues. Circular dichroism and tryptophan fluorescence indicate that these residues are substantially more constrained than similar residues in "classical" molten globules. Taken together, the data suggest a model for the A-state of equine lysozyme in which a more ordered core is surrounded by a less ordered but still compact polypeptide chain.

  • 24.
    Morozova-Roche, Ludmilla A
    et al.
    Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford.
    Jones, Jonathan A
    Noppe, Wim
    Dobson, Christopher M
    Independent nucleation and heterogeneous assembly of structure during folding of equine lysozyme1999In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 289, no 4, p. 1055-1073Article in journal (Refereed)
    Abstract [en]

    The refolding of equine lysozyme from guanidinium chloride has been studied using hydrogen exchange pulse labelling in conjunction with NMR spectroscopy and stopped flow optical methods. The stopped flow optical experiments indicate that extensive hydrophobic collapse occurs rapidly after the initiation of refolding. Pulse labelling experiments monitoring nearly 50 sites within the protein have enabled the subsequent formation of native-like structure to be followed in considerable detail. They reveal that an intermediate having persistent structure within three of the four helices of the alpha-domain of the protein is formed for the whole population of molecules within 4 ms. Subsequent to this event, however, the hydrogen exchange protection kinetics are complex and highly heterogeneous. Analysis of the results by fitting to stretched exponential functions shows that a series of other intermediates is formed as a consequence of the stepwise assembly of independently nucleated local regions of structure. In some molecules the next step in folding involves the stabilisation of the remaining helix in the alpha-domain, whilst in others persistent structure begins to form in the beta-domain. The formation of native-like structure throughout the beta-domain is itself heterogeneous, involving at least three kinetically distinguishable steps. Residues in loop regions throughout the protein attain persistent structure more slowly than regions of secondary structure. There is in addition evidence for locally misfolded regions of structure that reorganise on much longer timescales. The results reveal that the native state of the protein is generated by the heterogeneous assembly of a series of locally cooperative regions of structure. This observation has many features in common with the findings of recent theoretical simulations of protein folding.

  • 25.
    Nielsen, Søren B
    et al.
    Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
    Wilhelm, Kristina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vad, Brian
    Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Otzen, Daniel
    Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
    The interaction of equine lysozyme: oleic acid complexes with lipid membranes suggests a cargo off-loading mechanism2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 398, no 2, p. 351-361Article in journal (Refereed)
    Abstract [en]

    The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to alpha-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of alpha-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOA) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appears to be central to its biological action, similar to human alpha-lactalbumin made lethal to tumor cells (HAMLET). Here, we describe the interaction of ELOA with phospholipid membranes. Confocal scanning laser microscopy shows that ELOA, but not native EL, accumulates on the surface of giant unilamellar vesicles, without inducing significant membrane permeability. Quartz crystal microbalance with dissipation (QCM-D) data indicated an essentially non-disruptive binding of ELOA to supported lipid bilayers, leading to formation of highly dissipative and "soft" lipid membrane; at higher concentrations of ELOA, the lipid membrane desorbs from the surface probably as bilayer sheets of vesicles. This membrane rearrangement occurred to a similar extent when free oleic acid (OA) was added, but not when free OA was removed from ELOA by prior incubation with BSA, emphasizing the role of OA in this process. NMR data indicated an equilibrium between free and bound OA which shifts towards free OA as ELOA is progressively diluted indicating that OA is relatively loosely bound. Activity measurements together with fluorescence spectroscopy and circular dichroism suggested a conversion of ELOA toward a more native-like state on interaction with lipid membranes, although complete refolding was not observed. Altogether, these results suggest that ELOA may act as an OA carrier and facilitate OA transfer to the membrane. ELOA's properties illustrate that protein folding variants may possess specific functional properties distinct from the native protein. Abbreviations QCM-D, Quartz crystal microbalance with dissipation; CD, Circular dichroism; EL, equine lysozyme; ELOA, EL complex with oleic acid; OA, oleic acid, CSLM, Confocal scanning laser microscopy, Df, dissipation-frequency.

  • 26.
    Nilsson, Lina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pamrén, Annelie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Islam, Tohidul
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Golchin, Solmaz A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pettersson, Nina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Iakovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gharibyan, Anna L.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Transthyretin Interferes with Aβ Amyloid Formation by Redirecting Oligomeric Nuclei into Non-Amyloid Aggregates2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 17, p. 2722-2733Article in journal (Refereed)
    Abstract [en]

    The pathological Aβ aggregates associated with Alzheimer's disease follow a nucleation-dependent path of formation. A nucleus represents an oligomeric assembly of Aβ peptides that acts as a template for subsequent incorporation of monomers to form a fibrillar structure. Nuclei can form de novo or via surface-catalyzed secondary nucleation, and the combined rates of elongation and nucleation control the overall rate of fibril formation. Transthyretin (TTR) obstructs Aβ fibril formation in favor of alternative non-fibrillar assemblies, but the mechanism behind this activity is not fully understood. This study shows that TTR does not significantly disturb fibril elongation; rather, it effectively interferes with the formation of oligomeric nuclei. We demonstrate that this interference can be modulated by altering the relative contribution of elongation and nucleation, and we show how TTR's effects can range from being essentially ineffective to almost complete inhibition of fibril formation without changing the concentration of TTR or monomeric Aβ.

  • 27.
    Nord, Stefan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bylund, Göran O
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lövgren, J Mattias
    Wikström, P Mikael
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The RimP protein is important for maturation of the 30S ribosomal subunit2009In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 386, no 3, p. 742-753Article in journal (Refereed)
    Abstract [en]

    The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (DeltarimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 degrees C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.

  • 28.
    Näsvall, Joakim S
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nilsson, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The ribosomal grip of the peptidyl-tRNA is critical for reading frame maintenance.2009In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 385, no 2, p. 350-367Article in journal (Refereed)
    Abstract [en]

    If a ribosome shifts to an alternative reading frame during translation, the information in the message is usually lost. We have selected mutants of Salmonella typhimurium with alterations in tRNA(cmo5UGG)(Pro) that cause increased frameshifting when present in the ribosomal P-site. In 108 such mutants, two parts of the tRNA molecule are altered: the anticodon stem and the D-arm, including its tertiary interactions with the variable arm. Some of these alterations in tRNA(cmo5UGG)(Pro) are in close proximity to ribosomal components in the P-site. The crystal structure of the 30S subunit suggests that the C-terminal end of ribosomal protein S9 contacts nucleotides 32-34 of peptidyl-tRNA. We have isolated mutants with defects in the C-terminus of S9 that induce +1 frameshifting. Combinations of changes in tRNA(cmo5UGG)(Pro) and S9 suggest that an interaction occurs between position 32 of the peptidyl-tRNA and the C-terminal end of S9. Together, our results suggest that the cause of frameshifting is an aberrant interaction between the peptidyl-tRNA and the P-site environment. We suggest that the "ribosomal grip" of the peptidyl-tRNA is pivotal for maintaining the reading frame.

  • 29.
    Olofsson, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Hansson, Sebastian
    Umeå University, Faculty of Science and Technology, Chemistry.
    Hedberg, Linda
    Logan, Derek T
    Oliveberg, Mikael
    Folding of S6 structures with divergent amino acid composition: Pathway flexibility within partly overlapping foldons2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 365, no 1, p. 237-48Article in journal (Refereed)
    Abstract [en]

    Studies of circular permutants have demonstrated that the folding reaction of S6 from Thermus thermophilus (S6(T)) is malleable and responds in an ordered manner to changes of the sequence separation between interacting residues: the S6(T) permutants retain a common nucleation pattern in the form of a two-strand-helix motif that can be recruited from different parts of the structure. To further test the robustness of the two-strand-helix nucleus we have here determined the crystal structure and folding reaction of an evolutionary divergent S6 protein from the hyperthermophilic bacterium Aquifex aeolicus (S6(A)). Although the overall topology of S6(A) is very similar to that of S6(T) the architecture of the hydrophobic core is radically different by containing a large proportion of stacked Phe side-chains. Despite this disparate core composition, the folding rate constant and the kinetic m values of S6(A) are identical to those of S6(T). The folding nucleus of S6(A) is also found to retain the characteristic two-strand-helix motif of the S6(T) permutants, but with a new structural emphasis. The results suggest that the protein folding reaction is linked to topology only in the sense that the native-state topology determines the repertoire of accessible nucleation motifs. If the native structure allows several equivalent ways of recruiting a productive nucleus the folding reaction is free to redistribute within these topological constraints.

  • 30. Roper, D I
    et al.
    Subramanya, H S
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wigley, D B
    Preliminary crystallographic analysis of 4-oxalocrotonate tautomerase reveals the oligomeric structure of the enzyme.1994In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 243, no 4Article in journal (Refereed)
    Abstract [en]

    Crystals of recombinant 4-oxalocrotonate tautomerase from Pseudomonas sp. strain CF600 have been obtained in a form suitable for X-ray analysis. The enzyme is a highly efficient catalyst and is unusual in that it consists of subunits of only 62 amino acids. It crystallises in the triclinic space group, P1, with unit cell dimensions a = 39.6 A, b = 51.5 A, c = 51.6 A, alpha = 60.0 degrees, beta = 81.4 degrees, gamma = 69.6 degrees. The crystals diffract to beyond 1.9 A resolution and are stable to irradiation with X-rays. Preliminary crystallographic data are not consistent with the previously suggested pentameric structure, but indicate that the complex is in fact a hexamer with 32 symmetry.

  • 31.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Thomas, C M
    Analysis of the trfA region of broad host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products.1984In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 175, no 3Article in journal (Refereed)
    Abstract [en]

    Broad host-range plasmid RK2 is a member of the Escherichia coli incompatibility group P. Unlike most other groups of plasmids, members of the P group are capable of efficient transfer between and maintenance in most gram-negative bacterial species. It is of interest whether this broad host-range results from differences between the mechanism of replication of broad and narrow host-range plasmids. The regions of RK2 required for replication in E. coli have previously been defined as an origin of vegetative replication, oriVRK2 , and a gene, trfA , specifying a positively required trans-acting product. In this study Tn1723 transposon insertions have been used to map the trfA gene and determine its functional gene product. The Tn1723 insertions define the outer limits of the gene, a promoter region, a "leader" region not essential for trfA activity and a coding region. Three polypeptides of 13 X 10(3), 43 X 10(3) and 32 X 10(3) molecular weight are produced from this region and the production of a 32 X 10(3) Mr polypeptide is shown to be correlated with trfA activity in E. coli. Analysis of polypeptides produced from transposon insertion derivatives in which all but 35 base-pairs of inserted DNA is deleted, along with the effect of these insertions on trfA activity, suggest that the 43 X 10(3) and 32 X 10(3) Mr polypeptide coding sequences overlap in the same reading frame and that all three polypeptides (13 X 10(3), 32 X 10(3) and 43 X 10(3) Mr) may be translated from the same initial transcript.

  • 32.
    Song, Tianyan
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sabharwal, Dharmesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    VrrA mediates Hfq-dependent regulation of OmpT synthesis in Vibrio cholerae2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 400, no 4, p. 682-688Article in journal (Refereed)
    Abstract [en]

    OmpT, an outer membrane porin of Vibrio cholerae, is tightly regulated by the organism in response to different environments. Two transcriptional regulators, cAMP receptor protein (CRP) and ToxR, compete at the ompT promoter region. CRP activates ompT transcription by a loop-forming mechanism, while ToxR functions as an antiactivator and repressor, depending on its interplay with CRP. VrrA, a 140-nt small noncoding RNA in V. cholerae, is controlled by the alternative sigma factor sigma(E). We have demonstrated previously that VrrA represses ompA translation by base-pairing with the 5’ region of the mRNA, thereby affecting the release of outer membrane vesicles and modulating the colonization ability of V. cholerae. In this study, we demonstrate that VrrA RNA represses ompT translation by base-pairing with the 5’ region of the mRNA and that regulation requires the RNA chaperone protein Hfq. These results add new insight into the regulation of OmpT. In addition to pH/temperature signals via the ToxR regulon and carbon source signals via the cAMP-CRP complex, OmpT is further regulated by signals received via the sigma(E) regulon through VrrA.

  • 33. Teixeira, Pedro F.
    et al.
    Masuyer, Geoffrey
    Pinho, Catarina M.
    Branca, Rui M. M.
    Kmiec, Beata
    Wallin, Cecilia
    Wärmländer, Sebastian K. T. S.
    Berntsson, Ronnie P. -A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ankarcrona, Maria
    Gräslund, Astrid
    Lehtiö, Janne
    Stenmark, Pål
    Glaser, Elzbieta
    Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 3, p. 348-362Article in journal (Refereed)
    Abstract [en]

    Proteolysis plays an important role in mitochondrial biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLNE475Q in complex with the products of neurotensin cleavage at 2.7 Å revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-β peptide, Aβ1–40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aβ35–40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.

  • 34.
    Wallgren, Marcus
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pylypenko, Olena
    Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, 44202 Dortmund, Germany.
    Mikaelsson, Therese
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rak, Alexey
    Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, 44202 Dortmund, Germany.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 379, no 4, p. 845-858Article in journal (Refereed)
    Abstract [en]

    Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16meso) and hyperthermophilic (S16thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔCp0) of 6.4 and 3.3 kJ mol− 1 K− 1, respectively. A strong linear correlation between ΔCp0 and melting temperature (Tm) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16meso folds through an apparent two-state model, whereas S16thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16thermo is more compact relative to S16meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16thermo that appears to account for the large difference in quantified ΔCp0 values and, in turn, parts of the observed extreme thermal stability of S16thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.

  • 35.
    Wikström, P M
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Byström, Anders S
    Umeå University, Faculty of Medicine, Molecular Biology. Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Medicine, Molecular Biology.
    Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli1988In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 203, no 1, p. 141-152Article in journal (Refereed)
    Abstract [en]

    The trmD operon of Escherichia coli encodes the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and a 21,000 Mr protein of unknown function. Here we demonstrate that, in contrast to the expression of other ribosomal protein operons, the amount of trmD operon mRNA and the rate of synthesis of the proteins encoded by the operon respond to increased gene dosage. The steady-state level of the mRNA was about 18 times higher, and the relative rate of synthesis of the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and the 21,000 Mr protein was 15, 9, 25 and 23 times higher, respectively, in plasmid-containing cells than in plasmid-free cells. Overproduced tRNA(m1G37)methyltransferase and 21,000 Mr protein were as stable as E. coli total protein, whereas the two ribosomal proteins were degraded to a large extent. The steady-state amount of S16 and L19 in the plasmid-containing cells exceeded that in plasmid-free cells by threefold and twofold, respectively. No significant effect on the synthesis of the trmD operon proteins from the chromosomally located genes was observed when parts of the operon were expressed on different plasmids. Taken together, these results suggest that the expression of the trmD operon is not subject to transcriptional or translational feedback regulation, and demonstrate that not all ribosomal protein operons are regulated in the same manner. We propose that ribosomal protein operons that do not encode proteins that bind directly to rRNA are not under autogenous control. Metabolic regulation at the transcriptional level and protein degradation are plausible mechanisms for the control of expression of such operons.

  • 36.
    Wikström, P
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    O'Neill, E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ng, L C
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation.2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 314, no 5Article in journal (Refereed)
    Abstract [en]

    The transcriptional promoting activity of DmpR is under the strict control of its aromatic effector ligands that are bound by its regulatory N-terminal domain. The positive control function of DmpR resides within the central C-domain that is highly conserved among activators of sigma(54)-RNA polymerase. The C-domain mediates ATP hydrolysis and interaction with sigma(54)-RNA polymerase that are essential for open-complex formation and thus initiation of transcription. Wild-type and loss-of-function derivatives of DmpR, which are defective in distinct steps in nucleotide catalysis, were used to address the consequences of nucleotide binding and hydrolysis with respect to the multimeric state of DmpR and its ability to promote in vitro transcription. Here, we show that DmpR derivatives deleted of the regulatory N-terminal domain undergo an aromatic-effector independent ATP-binding triggered multimerisation as detected by cross-linking. In the intact protein, however, aromatic effector activation is required before ATP-binding can trigger an apparent dimer-to-hexamer switch in subunit conformation. The data suggest a model in which the N-terminal domain controls the transcriptional promoting property of DmpR by constraining ATP-mediated changes in its oligomeric state. The results are discussed in the light of recent mechanistic insights from the AAA(+) superfamily of ATPases that utilise nucleotide hydrolysis to restructure their substrates.

  • 37. Wiley, David J
    et al.
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Schesser, Kurt
    Induction of the Yersinia type 3 secretion system as an all-or-none phenomenon2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 373, no 1, p. 27-37Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia spp. possess a protein secretion system, designated as type 3, that plays a clear role in promoting their survival vis-à-vis the macrophage. Inductive expression of the Yersinia type 3 secretion system (T3SS), triggered either by host cell contact, or, in the absence of host cells, by a reduction in extracellular calcium ion levels, is accompanied by a withdrawal from the bacterial division cycle. Here, we analyzed Ca2+-dependent induction of the T3SS at the single-cell level to understand how Yersinia coordinates pro-survival and growth-related activities. We utilized a novel high-throughput quantitative microscopy approach as well as flow cytometry to determine how Ca2+ levels, T3SS expression, and cellular division are interrelated. Our analysis showed that there is a high degree of homogeneity in terms of T3SS expression levels among a population of Y. pseudotuberculosis cells following the removal of Ca2+, and that T3SS expression appears to be independent of the cellular division cycle. Unexpectedly, our analysis showed that Ca2+ levels are inversely related to the initiation of inductive T3SS expression, and not to the intensity of activation once initiated, thus providing a basis for the seemingly graded response of T3SS activation observed in bulk-level analyses. The properties of the system described here display both similarities to and differences from that of the lac operon first described 50 years ago by Novick and Weiner.

  • 38.
    Åström, Stefan U
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Medicine, Molecular Biology.
    Byström, Anders S
    Umeå University, Faculty of Medicine, Molecular Biology. Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    The yeast initiator tRNAMet can act as an elongator tRNA(Met) in vivo1993In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 233, no 1, p. 43-58Article in journal (Refereed)
    Abstract [en]

    Saccharomyces cerevisiae uses two different methionine accepting tRNAs during protein synthesis. One, tRNA(iMet), is used exclusively during the initiation of translation whereas the other, tRNA(mMet), is used during the elongation of translation. To study the unique features of each methionine tRNA species, we constructed yeast strains with null alleles of the five elongator methionine tRNA (EMT) genes and strains with null alleles of the four initiator methionine tRNA (IMT) genes, respectively. Consequently, growth of these strains was dependent either on a tRNA(mMet) or a tRNA(iMet), respectively, encoded from a plasmid-derived gene. For both null mutants, the plasmid carrying the wild-type gene can be selected against and exchanged for another plasmid derived EMT or IMT gene (wild-type or mutant). A high gene dosage of the wild-type IMT gene could restore growth to the elongator-depleted strain. However, wild-type EMT genes in a high gene dosage never restored growth of the initiator depleted strain. Thus, the elongator tRNA(Met) is much more restricted to participate in the initiation of translation than the initiator tRNA(Met) is restricted to participate in the elongation process. Using the two null mutants, we have identified tRNA(mMet) mutants, which show reduced elongator activity, and tRNA(iMet) mutants, with improved elongator activity in the elongator depleted strain. Also, tRNA(mMet) mutants that function as an initiator tRNA in the initiator depleted strain were identified. From this mutant analysis, we showed that the conserved U/rT at position 54 of the elongator tRNA(Met) is an important determinant for an elongator tRNA. The most important determinant for an initiator was shown to be the acceptor stem and especially the conserved A1.U72 base-pair. Mutant tRNAs, with reduced activity in either process, were investigated for enhanced activity during overproduction of the alpha and beta-subunits of the eukaryotic initiation factor 2 (eIF-2) or the eukaryotic elongation factor 1 alpha (eEF-1 alpha). The data suggest that the U/rT of the elongator at position 54 is important for eEF-1 alpha recognition and that the acceptor stem of the initiator is important for eIF-2 recognition.

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