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  • 451.
    Keskin, Isil
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    SOD, ORF and ALS: On the role of SOD1 and C9ORF72 in the pathogenesis of ALS2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset degeneration of upper and lower motor neurons. Symptoms begin focally in one muscle and then spread contiguously, resulting in progressive paralysis and death from respiratory failure. Hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause, however, mutations in SOD1 were the first identified and are found in 1-9% of patients. Misfolded SOD1 aggregates in the CNS are hallmarks of ALS associated with SOD1 mutations. However, accumulation of misfolded or aggregated SOD1 protein has also been reported in sporadic and familial ALS without SOD1 mutations, suggesting that wild-type SOD1 could play a role in ALS pathology in general.

    The aims of this thesis are: 1) To describe the resulting disease phenotype and specific characteristics of the SOD1 protein carrying the stable disease- associated mutation L117V. 2) To set up cell-based in vitro models to study the mechanisms of SOD1 misfolding and aggregation under physiologically relevant expression levels. 3) To compare SOD1 activity in patient-derived samples and screen for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations.

    1) We identified a novel L117V SOD1 mutant in two families of Syrian origin that co-segregated with the disease. This mutation was associated with slow disease progression, reduced penetrance and a uniform phenotype. The L117V mutant protein was indistinguishable from wild-type SOD1 in terms of stability, dismutation activity and misfolding in patient-derived cell lines.

    2) We established patient-derived fibroblast and iPSC-MN lines expressing mutant SOD1 at physiological levels as in vitro models to study misfolding and aggregation of SOD1. We investigated the effects of several cellular pathway disturbances on SOD1 misfolding. Misfolded SOD1 was increased by inhibition of the ubiquitin-proteasome pathway in fibroblasts derived from both patients and controls. An age-related decline in proteasome activity could contribute to the late onset of ALS.

    Next, we studied the effects of low oxygen tension on misfolding and aggregation of SOD1 in patient-derived cells. Low O2 tensions were found to markedly increase C57-C146 disulphide reduction, misfolding and aggregation of SOD1. Importantly, the largest effects were detected in iPSC-MNs. This suggests that motor neurons are specifically vulnerable to misfolding and aggregation of SOD1 under low O2 tension.

    3) We compared the enzymatic activity of SOD1 in blood samples from a large number of ALS patients and controls. We screened for potential underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. No aberrations in copy number, other large structural changes in introns and exons or intronic mutations in the 30-50 bp flanking the exons were found in the 142 outliers, with either very low or very high SOD1 dismutation activities. However, hemoglobinopathies, including thalassemias and iron deficiency anemia, were associated with high SOD1/mg Hb ratios. Erythrocytes from patients with destabilizing SOD1 mutations showed half the normal activity. There were no significant differences in SOD1 activity between control individuals and ALS patients without a coding SOD1 mutation, or carriers of TBK1 mutations or the hexanucleotide repeat expansion in C9ORF72. Our result suggests that SOD1 enzymatic activity is not associated with the disease in non-SOD1 mutation ALS.

  • 452.
    Keskin, Isil
    et al.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Birve, Anna
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Berdynski, Mariusz
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Hjertkvist, Karin
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Rofougaran, Reza
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Nilsson, Torbjörn K.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Glass, Jonathan D.
    Marklund, Stefan L.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Andersen, Peter M.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Comprehensive analysis to explain reduced or increased SOD1 enzymatic activity in erythrocytes in ALS patients and their relativesManuscript (preprint) (Other academic)
    Abstract [en]

    Our objective was to in blood samples from 3723 individuals including ALS patients without a coding SOD1 mutation and 372 control individuals characterize stabilities of mutant SOD1s, compare SOD1 enzymatic activities between patients with different genetic causes of ALS, and search for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. Erythrocyte SOD1 enzymatic activities normalized to hemoglobin content were determined. Coding SOD1 sequences were analyzed by Sanger sequencing, copy number variations by fragment length analysis and by TaqMan Assay. Hemoglobin disorders were searched for. Of the 46 SOD1 mutations found, ¾ caused severe destabilization of the mutant protein but in ¼ SOD1 was essentially physically stable. Mutations producing structural changes all caused halved SOD activities. There were no differences in SOD1 activities between controls and patients without any detected SOD1 mutations or patients with C9ORF72HRE or TBK1 mutations. In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Also, no uncommon variants in exon-flanking sequences were detected. Thalassemias and iron deficiency anemia were associated with increased SOD1 activity/hemoglobin ratios. In conclusion, adjunct erythrocyte SOD activity analysis is of value to signal the presence of exon and splice-site-intron mutations that influence the SOD1 structure.

  • 453.
    Keskin, Isil
    et al.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Forsgren, Elin
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Andersen, Peter M.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Lange, Dale J.
    Synofzik, Matthis
    Nordström, Ulrika
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Zetterström, Per
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Marklund, Stefan L.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Gilthorpe, Jonathan D.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Low oxygen tension induces misfolding and aggregation of superoxide dismutase in ALS patient-derived motor neuronsManuscript (preprint) (Other academic)
  • 454.
    Keskin, Isil
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Forsgren, Elin
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Lehmann, Manuela
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Andersen, Peter M.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Brännström, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Lange, Dale J.
    Synofzik, Matthis
    Nordström, Ulrika
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Zetterström, Per
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Marklund, Stefan L.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Gilthorpe, Jonathan D.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    The molecular pathogenesis of superoxide dismutase 1-linked ALS is promoted by low oxygen tension2019In: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 138, no 1, p. 85-101Article in journal (Refereed)
    Abstract [en]

    Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS). Disease pathogenesis is linked to destabilization, disorder and aggregation of the SOD1 protein. However, the non-genetic factors that promote disorder and the subsequent aggregation of SOD1 have not been studied. Mainly located to the reducing cytosol, mature SOD1 contains an oxidized disulfide bond that is important for its stability. Since O2 is required for formation of the bond, we reasoned that low O2 tension might be a risk factor for the pathological changes associated with ALS development. By combining biochemical approaches in an extensive range of genetically distinct patient-derived cell lines, we show that the disulfide bond is an Achilles heel of the SOD1 protein. Culture of patient-derived fibroblasts, astrocytes, and induced pluripotent stem cell-derived mixed motor neuron and astrocyte cultures (MNACs) under low oxygen tensions caused reductive bond cleavage and increases in disordered SOD1. The effects were greatest in cells derived from patients carrying ALS-linked mutations in SOD1. However, significant increases also occurred in wild-type SOD1 in cultures derived from non-disease controls, and patients carrying mutations in other common ALS-linked genes. Compared to fibroblasts, MNACs showed far greater increases in SOD1 disorder and even aggregation of mutant SOD1s, in line with the vulnerability of the motor system to SOD1-mediated neurotoxicity. Our results show for the first time that O2 tension is a principal determinant of SOD1 stability in human patient-derived cells. Furthermore, we provide a mechanism by which non-genetic risk factors for ALS, such as aging and other conditions causing reduced vascular perfusion, could promote disease initiation and progression.

  • 455.
    Khoshnood, Behzad
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Function and targets of the Urm1/Uba4 conjugation machinery in Drosophila melanogaster2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Posttranslational modification (PTM) of proteins is essential to maintain homeostasis and viability in all eukaryotic cells. Hence, besides the sequence and 3D folding of a polypeptide, modification by multiple types of PTMs, ranging from small molecular groups to entire protein modules, adds another layer of complexity to protein function and regulation. The ubiquitin-like modifiers (UBLs) are such a group of evolutionary conserved protein modifiers, which by covalently conjugating to target proteins can modulate the subcellular localization and activity of their targets. One example of such a UBL, is the Ubiquitin related modifier 1 (Urm1). Since its discovery in 2000, Urm1 has been depicted as a dual function protein, which besides acting as a PTM, in addition functions as a sulfur carrier during the thio-modification of a specific group of tRNAs. Due to this dual capacity, Urm1 is considered as the evolutionary ancestor of the entire UBL family. At present, it is well established that Urm1, with help of its dedicated E1 enzyme Uba4/MOCS3, conjugates to multiple target proteins (urmylation) and that Urm1 thus plays important roles in viability and the response against oxidative stress.

    The aim of this thesis has been to, for the first time, investigate the role of Urm1 and Uba4 in a multicellular organism, utilising a multidisciplinary approach that integrates Drosophila genetics with classical biochemical assays and proteomics. In Paper I, we first characterized the Drosophila orthologues of Urm1 (CG33276) and Uba4 (CG13090), verified that they interact physically as well as genetically, and that they together can induce urmylation in the fly. By subsequently generating an Urm1 null Drosophila mutant (Urm1n123), we established that Urm1 is essential for viability and that flies lacking Urm1 are resistant to oxidative stress. Providing a molecular explanation for this phenotype, we demonstrated an involvement of Urm1 in the regulation of JNK signaling, including the transcription of the cytoprotective genes Jafrac1 and gstD1. Besides the resistance to oxidative stress, we have moreover (Manuscript IV) made an in-depth investigation of another phenotype displayed by Urm1n123 mutants, an overgrowth of third instar larval neuromuscular junctions (NMJs), a phenotype which is shared also with mutants lacking Uba4 (Uba4n29).

    To increase the understanding of Urm1 in the fly, we next employed a proteomics-based approach to identify candidate Urm1 target proteins (Paper II). Using this strategy, we identified 79 Urm1-interacting proteins during three different stages of fly development. Of these, six was biochemically confirmed to interact covalently with Urm1, whereas one was found to be associated with Urm1 by non-covalent means. In Manuscript III, we additionally identified the virally encoded oncogene Tax as a target of Urm1, both in Drosophila tissues and mammalian cell lines. In this study, we established a strong correlation between Tax urmylation and subcellular localization, and that Urm1 promoted a cytoplasmic accumulation and enhanced signalling activity of Tax, with implications for a potential role of Urm1 in Tax-induced oncogenesis.

    Taken together, this thesis provides a basic understanding of the potential roles and targets of Urm1 in a multicellular organism. The four studies included cover different aspects of Urm1 function and clearly points towards a highly dynamic role of protein urmylation in fly development, as well as in adult life.

  • 456.
    Khoshnood, Behzad
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Dacklin, Ingrid
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Grabbe, Caroline
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 9, article id e0185611Article in journal (Refereed)
    Abstract [en]

    By covalently conjugating to target proteins, ubiquitin-like modifiers (UBLs) act as important regulators of target protein localization and activity, thereby playing a critical role in the orchestration of cellular biology. The most ancient and one of the least studied UBLs is Urm1, a dual-function protein that in parallel to performing similar functions as its prokaryotic ancestors in tRNA modification, also has adopted the capacity to conjugate to cellular proteins analogous to ubiquitin and other UBL modifiers. In order to increase the understanding of Urm1 and its role in multicellular organisms, we have used affinity purification followed by mass spectrometry to identify putative targets of Urm1 conjugation (urmylation) at three developmental stages of the Drosophila melanogaster lifecycle. Altogether we have recovered 79 Urm1-interacting proteins in Drosophila, which include the already established Urm1 binding partners Prx5 and Uba4, together with 77 candidate urmylation targets that are completely novel in the fly. Among these, the majority was exclusively identified during either embryogenesis, larval stages or in adult flies. We further present biochemical evidence that four of these proteins are covalently conjugated by Urm1, whereas the fifth verified Urm1-binding protein appears to interact with Urm1 via non-covalent means. Besides recapitulating the previously established roles of Urm1 in tRNA modification and during oxidative stress, functional clustering of the newly identified Urm1-associated proteins further positions Urm1 in protein networks that control other types of cellular stress, such as immunological threats and DNA damage. In addition, the functional characteristics of several of the candidate targets strongly match the phenotypes displayed by Urm1(n123) null animals, including embryonic lethality, reduced fertility and shortened lifespan. In conclusion, this identification of candidate targets of urmylation significantly increases the knowledge of Urm1 and presents an excellent starting point for unravelling the role of Urm1 in the context of a complex living organism.

  • 457.
    Khoshnood, Behzad
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University.
    Dacklin, Ingrid
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grabbe, Caroline
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Deciphering a novel role of the Urm1/Uba4 conjugation machinery for Neuromuscular Junction (NMJ) development in Drosophila melanogasterManuscript (preprint) (Other academic)
    Abstract [en]

    In Drosophila melanogaster, development of the part of the peripheral nervous system that is dedicated to orchestrate locomotion, relies on an intricate interplay between the motor neurons that emanate from the ventral nerve cord, and the body wall musculature that they are destined to innervate. A rather large array of cytoskeletal and signaling proteins are implicated in the formation and growth of the synapses, where motor neurons contact the musculature, specifically at structures known as neuromuscular junctions (NMJs). Master regulators of NMJ development and growth include the WNT, BMP/TGFβ, MAPK, PI3K and JNK signaling pathways. Here we describe a novel role of the ubiquitin-like molecule (UBL) Urm1 (Ubiquitin-related modifier 1) in the regulation of NMJ formation. Specifically, we show that Drosophila Urm1n123 null mutants, as well as flies deficient of the dedicated Urm1 E1 activating enzyme Uba4, Uba4n29, display a significant NMJ overgrowth in third instar larvae, and that Urm1 and Uba4 interact genetically in this process. By utilizing the UAS/GAL4 system, we further provide evidence that Urm1 appears to act in both pre- and post-synaptic tissues, but rescue experiments emphasize a primary role of Urm1 in presynaptic motor neurons. In keeping with the previously established link between loss of Urm1 and excessive activation of JNK signaling, we can conclude that there is a strong genetic interaction between Urm1 and Drosophila JNK pathway components also during NMJ formation. Taken together, we have identified a role of the Urm1/Uba4 conjugation machinery in the regulation of NMJ development and growth, putatively induced by enhanced pre-synaptic activation of the JNK pathway.

  • 458.
    Khoshnood, Behzad
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Dacklin, Ingrid
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grabbe, Caroline
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Urm1: an essential regulator of JNK signaling and oxidative stress in Drosophila melanogaster2016In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 73, no 9, p. 1939-1954Article in journal (Refereed)
    Abstract [en]

    Ubiquitin-related modifier 1 (Urm1) is a ubiquitin-like molecule (UBL) with the dual capacity to act both as a sulphur carrier and posttranslational protein modifier. Here we characterize the Drosophila melanogaster homologues of Urm1 (CG33276) and its E1 activating enzyme Uba4 (CG13090), and show that they function together to induce protein urmylation in vivo. Urm1 conjugation to target proteins in general, and to the evolutionary conserved substrate Peroxiredoxin 5 (Prx5) specifically, is dependent on Uba4. A complete loss of Urm1 is lethal in flies, although a small number of adult zygotic Urm1 (n123) mutant escapers can be recovered. These escapers display a decreased general fitness and shortened lifespan, but in contrast to their S. cerevisiae counterparts, they are resistant to oxidative stress. Providing a molecular explanation, we demonstrate that cytoprotective JNK signaling is increased in Urm1 deficient animals. In agreement, molecular and genetic evidence suggest that elevated activity of the JNK downstream target genes Jafrac1 and gstD1 strongly contributes to the tolerance against oxidative stress displayed by Urm1 (n123) null mutants. In conclusion, Urm1 is a UBL that is involved in the regulation of JNK signaling and the response against oxidative stress in the fruit fly.

  • 459.
    Kieselbach, Thomas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cheregi, Otilia
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Green, Beverley R.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Proteomic analysis of the phycobiliprotein antenna of the cryptophyte alga Guillardia theta cultured under different light intensities2018In: Photosynthesis Research, ISSN 0166-8595, E-ISSN 1573-5079, Vol. 135, no 1–3, p. 149-163Article in journal (Refereed)
    Abstract [en]

    Plants and algae have developed various light-harvesting mechanisms for optimal delivery of excitation energy to the photosystems. Cryptophyte algae have evolved a novel soluble light-harvesting antenna utilizing phycobilin pigments to complement the membrane-intrinsic Chl a/c-binding LHC antenna. This new antenna consists of the plastid-encoded β-subunit, a relic of the ancestral phycobilisome, and a novel nuclear-encoded α-subunit unique to cryptophytes. Together, these proteins form the active α1β·α2β-tetramer. In all cryptophyte algae investigated so far, the α-subunits have duplicated and diversified into a large gene family. Although there is transcriptional evidence for expression of all these genes, the X-ray structures determined to date suggest that only two of the α-subunit genes might be significantly expressed at the protein level. Using proteomics, we show that in phycoerythrin 545 (PE545) of Guillardia theta, the only cryptophyte with a sequenced genome, all 20 α-subunits are expressed when the algae grow under white light. The expression level of each protein depends on the intensity of the growth light, but there is no evidence for a specific light-dependent regulation of individual members of the α-subunit family under the growth conditions applied. GtcpeA10 seems to be a special member of the α-subunit family, because it consists of two similar N- and C-terminal domains, which likely are the result of a partial tandem gene duplication. The proteomics data of this study have been deposited to the ProteomeXchange Consortium and have the dataset identifiers PXD006301 and 10.6019/PXD006301.

  • 460. Kim, Maria V
    et al.
    Kasakov, Alexei S
    Seit-Nebi, Alim S
    Marston, Steven B
    Gusev, Nikolai B
    Structure and properties of K141E mutant of small heat shock protein HSP22 (HspB8, H11) that is expressed in human neuromuscular disorders.2006In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 454, no 1Article in journal (Refereed)
    Abstract [en]

    Some properties of the K141E mutant of human HSP22 that is expressed in distal hereditary motor neuropathy were investigated. This mutation slightly decreased intrinsic fluorescence of HSP22 and induced changes in the far UV CD spectra that correlate with increase of disordered structure. Destabilized K141E mutant was more susceptible to trypsinolysis than the wild type protein. Mutation K141E did not significantly affect the hydrophobic properties measured by bis-ANS binding and did not affect the quaternary structure of HSP22. With insulin as a substrate the chaperone-like activity of K141E mutant and the wild type protein were similar. However with alcohol dehydrogenase and rhodanese the chaperone-like activity of K141E mutant was remarkably lower than the corresponding activity of the wild type protein. It is concluded that K141E mutation induces destabilization of HSP22 structure and probably by this means diminish the chaperone-like activity of HSP22 with certain protein substrates.

  • 461. Kim, Maria V
    et al.
    Seit-Nebi, Alim S
    Gusev, Nikolai B
    The problem of protein kinase activity of small heat shock protein Hsp22 (H11 or HspB8).2004In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 325, no 3Article in journal (Refereed)
    Abstract [en]

    The recently described protein denoted H11, Hsp22 or HspB8 seems to participate in regulation of proliferation, apoptosis, and cardiac hypertrophy. Mutation of Hsp22 causes distal motor neuropathy. Multitude action of Hsp22 is supposed to be due to its protein kinase and/or chaperone-like activities. There are many indirect evidences indicating that Hsp22 possesses intrinsic protein kinase activity. However, low homology to protein kinases, low extent of autophosphorylation, lack of significant protein kinase activity with commonly used substrates, and lack of information on stoichiometry, kinetics, and substrate specificity make the existence of intrinsic protein kinase activity of Hsp22 questionable. It is supposed that protein kinase activity ascribed to Hsp22 is due to contaminating protein kinases. Hsp22 is highly homologous to small heat shock proteins and effectively prevents aggregation of denatured protein both in vitro and in vivo. Therefore, it is supposed that chaperone-like activity is of great importance for Hsp22 functioning.

  • 462. Kim, Maria V
    et al.
    Seit-Nebi, Alim S
    Marston, Steven B
    Gusev, Nikolai B
    Some properties of human small heat shock protein Hsp22 (H11 or HspB8).2004In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 315, no 4Article in journal (Refereed)
    Abstract [en]

    Untagged recombinant human small heat shock protein with apparent molecular mass 22 kDa (Hsp22) was obtained in homogeneous state. Size exclusion chromatography and chemical crosslinking with dimethylsuberimidate indicate that Hsp22 forms stable dimers. Being highly susceptible to oxidation Hsp22 forms disulfide crosslinked dimers and poorly soluble high molecular mass oligomers. According to CD spectroscopy oxidation of Hsp22 results in disturbing of both secondary and tertiary structure. Hsp22 possesses a negligibly low autophosphorylation activity and under the conditions used is unable to phosphorylate casein or histone. Hsp22 effectively prevents heat-induced aggregation of yeast alcohol dehydrogenase and bovine liver rhodanese with chaperone activity comparable to that of recombinant human small heat shock protein with apparent molecular mass 20 kDa (Hsp20).

  • 463.
    Kindgren, Peter
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    The chloroplast talks: Insights into the language of the chloroplast in Arabidopsis2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The chloroplast originates from an endosymbiotic event 1.5 billion years ago, when a free living photosynthetic bacteria was engulfed by a eukaryotic host. The chloroplastic genome has through evolution lost many genes to the nuclear genome of the host. To coordinate the gene expression between the two genomes, plants have evolved two types of communication, nucleus-to-plastid (anterograde) and plastid-to-nucleus (retrograde) signalling. This thesis will focus on retrograde communication with emphasis on redox and tetrapyrrole mediated signalling.

    In this thesis, we establish the tetrapyrrole Mg-ProtoIX as an important retrograde negative regulator of nuclear encoded plastid proteins. We show that Mg-ProtoIX accumulates in both artificial and natural stress conditions, and that the accumulation is tightly correlated to regulation of nuclear gene expression. Using confocal microscopy, we could visualize Mg-ProtoIX in the cytosol during stress conditions. In addition, exogenously applied Mg-ProtoIX stayed in the cytosol and was enough to trigger a signal to the nucleus. The results presented here indicate that Mg-ProtoIX is transported out of the chloroplast to control nuclear gene expression. Mg-ProtoIX mediated repression of the nuclear gene, COR15a, occurs via the transcription factor HY5. HY5 is influenced by both plastid signals and the photoreceptors. Here, we show that photoreceptors are part of Mg-ProtoIX mediated signalling as well as excess light adaptation. We identified the blue light receptor, CRY1, as a light intensity sensor that partly utilizes HY5 in the high light response. To further understand the high light regulation of nuclear genes, we isolated a mutant with redox insensitive (rin) high light response. The rin2 mutant has a mutated plastid protein with unknown function. Characterization of the rin2 mutant revealed that the protein is important in regulating plastid gene expression as well as nuclear gene expression. The rin2 mutant is the first characterized rin mutant and could prove important in elucidating the cross-talk between redox mediated coordination between the plastid and the nuclear genome.

  • 464.
    Király, Kari
    et al.
    Department of Anatomy, University o Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Anatomy, University o Kuopio, Kuopio, Finland.
    Arokoski, Jari
    Department of Anatomy, University o Kuopio, Kuopio, Finland.
    Lapveteläinen, Tuomo
    Department of Anatomy, University o Kuopio, Kuopio, Finland.
    Tammi, Markku
    Department of Anatomy, University o Kuopio, Kuopio, Finland.
    Helminen, Heikki
    Department of Anatomy, University o Kuopio, Kuopio, Finland.
    Kiviranta, Ilkka
    Safranin O reduces loss of glycosaminoglycans from bovine articular cartilage during histological specimen preparation.1996In: The Histochemical Journal, ISSN 0018-2214, E-ISSN 1573-6865, Vol. 28, no 2, p. 99-107, article id 8737291Article in journal (Refereed)
    Abstract [en]

    The ability of Safranin O, added to fixation and decalcification solutions, to prevent the escape of glycosaminoglycans (GAGs) from small cartilage tissue blocks during histological processing of cartilage has been studied. GAGs in the fixatives and decalcifying solutions used and those remaining in the 1 mm3 cubes of cartilage were assayed biochemically. The quantity of GAGs remaining in the cartilage cubes were determined from Safranin O-stained sections using videomicroscopy or microspectrophotometry. A quantity (10.6%) of GAGs were lost during a conventional 4% buffered formaldehyde fixation (48 h) and a subsequent decalcification in 10% EDTA (12 days) at 4 degrees C. Roughly one-quarter of the total GAG loss occurred during the 48 h fixation, and three-quarters during the 12 days of decalcification. Inclusion of 4% formaldehyde in the decalcification fluid decreased the loss of GAGs to 6.2%. The presence of 0.5% Safranin O in the fixative reduced this loss to 3.4%. When 0.5% Safranin O was included in the fixative and 4% formaldehyde in the decalcification solution, Safranin O staining of the histological sections increased on average by 13.5%. After fixation in the presence of 0.5% Safranin O, there was no difference in the staining intensities when decalcification was carried out in the presence of either Safranin O or formaldehyde, or both. It took 24 h for Safranin O to penetrate into the deep zone of articular cartilage, warranting a fixation period of at least this long. In conclusion, the addition of Safranin O to the fixative and either Safranin O or formaldehyde in the following decalcification fluid, markedly reduces the loss of GAGs from small articular cartilage explants during histological processing. However, for immunohistochemical studies, Safranin O cannot be included in the processing solutions, because it may interfere.

  • 465. Kisand, Veljo
    et al.
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences2003In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 54, no 2, p. 183-191Article in journal (Refereed)
    Abstract [en]

    The phylogenetic affiliation of 91 operational taxonomic units, randomly sampled from three aquatic microcosm experiments, was investigated by two PCR based and one culture dependent method. The occurrence of multiple melting domains and poor coupling between Tin and DGGE retardation was demonstrated to cause poor resolution at the species level in PCR-DGGE analysis of microbial communities. We also showed that the problem of multiple melting domains was particularly prone for brackish water bacterioplankton in the Flavobacterium genus, providing characteristic band morphology for this genus. Banding patterns from DGGE analysis may therefore be misinterpreted in terms of the species richness in natural bacterial communities, when using commonly applied universal primers. (C) 2003 Elsevier Science B.V. All rights reserved.

  • 466. Kitamura, Aya
    et al.
    Nishimoto, Madoka
    Sengoku, Toru
    Shibata, Rie
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Grosjean, Henri
    Yokoyama, Shigeyuki
    Bessho, Yoshitaka
    Characterization and Structure of the Aquifex aeolicus Protein DUF752 A BACTERIAL tRNA-METHYLTRANSFERASE (MnmC2) FUNCTIONING WITHOUT THE USUALLY FUSED OXIDASE DOMAIN (MnmC1)2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 52, p. 43950-43960Article in journal (Refereed)
    Abstract [en]

    Post-transcriptional modifications of the wobble uridine (U34) of tRNAs play a critical role in reading NNA/G codons belonging to split codon boxes. In a subset of Escherichia coli tRNA, this wobble uridine is modified to 5-methylaminomethyluridine (mnm5U34) through sequential enzymatic reactions. Uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm5U34) by the MnmE-Mnm Genzyme complex. The cmnm5U34 is further modified to mnm5U by the bifunctional MnmC protein. In the first reaction, the FAD-dependent oxidase domain (MnmC1) converts cmnm5U into 5-aminomethyluridine (nm5U34), and this reaction is immediately followed by the methylation of the free amino group into mnm5U34 by the S-adenosylmethionine-dependent domain (MnmC2). Aquifex aeolicus lacks a bifunctional MnmC protein fusion and instead encodes the Rossmann-fold protein DUF752, which is homologous to the methyltransferase MnmC2 domain of Escherichia coli MnmC (26% identity). Here, we determined the crystal structure of the A. aeolicus DUF752 protein at 2.5 Å resolution, which revealed that it catalyzes the S-adenosylmethionine-dependent methylation of nm5U in vitro, to form mnm5U34 in tRNA. We also showed that naturally occurring tRNA from A. aeolicus contains the 5-mnm group attached to the C5 atom of U34. Taken together, these results support the recent proposal of an alternative MnmC1-independent shortcut pathway for producing mnm5U34 in tRNAs.

  • 467.
    Kleczkowski, Leszek A.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Decker, Daniel
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Sugar activation for production of nucleotide sugars as aubstrates for glycosyltransferases in plants2015In: Journal of Applied Glycoscience, ISSN 1344-7882, Vol. 62, no 2, p. 25-36Article, review/survey (Refereed)
    Abstract [en]

    In order to serve as a glycosyl donor, a sugar or a sugar derivative (e.g. GlcA) needs to be “activated” to a highly energetic state of a nucleotide-sugar. This activation requires the involvement of specific enzymes which produce NDP-sugars (or, in one case, NMP-sugar), using NTP or NDP as substrate. The present review provides concise survey of distinct plant nucleotide-sugar pyrophosphorylases (all using NTP as one of the substrates and differing in sugar specificity) as well as nucleotide-sugar phosphorylases and sucrose synthase (all using NDP as one of substrates). The pyrophosphorylases discussed include UGPase, USPase, UAGPase, AGPase, GMPase (VTC1), and FKGP, whereas phosphorylases include ADP-Glc phosphorylase and GDP-Gal phosphorylase (VTC2/VTC5). We also discuss the activation mechanism of 3-deoxy-D-manno-octulosonic acid (Kdo) by CKS, leading to the formation of a unique NMP-linked sugar (CMP-Kdo).

  • 468.
    Kleczkowski, Leszek A
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Decker, Daniel
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    UDP-sugar pyrophosphorylase: a new old mechanism for sugar activation2011In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 156, no 1, p. 3-10Article in journal (Refereed)
    Abstract [en]

    Recent developments in studies on properties and functions of UDP-sugar pyrophosphorylase (USPase) in metabolism are presented. The protein was characterized from plants and protozoans (Leishmania, Trypanosoma), but apparently it is also present in bacteria. In plants, USPase deficiency leads to male-sterility. USPase produces a variety of UDP-sugars and their analogs required for cell wall biosynthesis as well as for protein and lipid glycosylation, among other functions. Substrate specificity of USPases from different sources is reviewed, and their function/ structure properties are discussed, based on recent crystallization of the protein, with emphasis on common structural blueprint with some other pyrophosphorylases. Some strategies for future research on USPase are discussed.

  • 469.
    Kleczkowski, Leszek A.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Geisler, Matt
    Fitzek, Elisabeth
    Wilczynska, Malgorzata
    A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 439, p. 375-379Article, review/survey (Refereed)
    Abstract [en]

    Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

  • 470.
    Klemenčič, Marina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.
    Asplund-Samuelsson, Johannes
    Dolinar, Marko
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Phylogenetic Distribution and Diversity of Bacterial Pseudo-Orthocaspases Underline Their Putative Role in Photosynthesis2019In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 10, article id 293Article in journal (Refereed)
    Abstract [en]

    Orthocaspases are prokaryotic caspase homologs – proteases, which cleave their substrates after positively charged residues using a conserved histidine – cysteine (HC) dyad situated in a catalytic p20 domain. However, in orthocaspases pseudo-variants have been identified, which instead of the catalytic HC residues contain tyrosine and serine, respectively. The presence and distribution of these presumably proteolytically inactive p20-containing enzymes has until now escaped attention. We have performed a detailed analysis of orthocaspases in all available prokaryotic genomes, focusing on pseudo-orthocaspases. Surprisingly we identified type I metacaspase homologs in filamentous cyanobacteria. While genes encoding pseudo-orthocaspases seem to be absent in Archaea, our results show conservation of these genes in organisms performing either anoxygenic photosynthesis (orders Rhizobiales, Rhodobacterales, and Rhodospirillales in Alphaproteobacteria) or oxygenic photosynthesis (all sequenced cyanobacteria, except Gloeobacter, Prochlorococcus, and Cyanobium). Contrary to earlier reports, we were able to detect pseudo-orthocaspases in all sequenced strains of the unicellular cyanobacteria Synechococcus and Synechocystis. In silico comparisons of the primary as well as tertiary structures of pseudo-p20 domains with their presumably proteolytically active homologs suggest that differences in their amino acid sequences have no influence on the overall structures. Mutations therefore affect most likely only the proteolytic activity. Our data provide an insight into diversification of pseudo-orthocaspases in Prokaryotes, their taxa-specific distribution, and allow suggestions on their taxa-specific function.

  • 471.
    Klemenčič, Marina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural and functional diversity of caspase homologues in non-metazoan organisms2018In: Protoplasma, ISSN 0033-183X, E-ISSN 1615-6102, Vol. 255, no 1, p. 387-397Article, review/survey (Refereed)
    Abstract [en]

    Caspases, the proteases involved in initiation and execution of metazoan programmed cell death, are only present in animals, while their structural homologues can be found in all domains of life, spanning from simple prokaryotes (orthocaspases) to yeast and plants (metacaspases). All members of this wide protease family contain the p20 domain, which harbours the catalytic dyad formed by the two amino acid residues, histidine and cysteine. Despite the high structural similarity of the p20 domain, metacaspases and orthocaspases were found to exhibit different substrate specificities than caspases. While the former cleave their substrates after basic amino acid residues, the latter accommodate substrates with negative charge. This observation is crucial for the re-evaluation of non-metazoan caspase homologues being involved in processes of programmed cell death. In this review, we analyse the structural diversity of enzymes containing the p20 domain, with focus on the orthocaspases, and summarise recent advances in research of orthocaspases and metacaspases of cyanobacteria, algae and higher plants. Although caspase homologues were initially proposed to be involved in execution of cell death, accumulating evidence supports the role of metacaspases and orthocaspases as important contributors to cell homeostasis during normal physiological conditions or cell differentiation and ageing.

  • 472.
    Kolmert, Johan
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå ; Global Safety Assessment, AstraZeneca R&D, Södertälje ; Department of Analytical Chemistry, Stockholm University.
    Forngren, B.
    Lindberg, J.
    Öhd, J.
    Åberg, K. M.
    Nilsson, G.
    Moritz, T.
    Nordström, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    A quantitative LC/MS method targeting urinary 1-methyl-4-imidazoleacetic acid for safety monitoring of the global histamine turnover in clinical studies2014In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 6, p. 1751-1762Article in journal (Refereed)
    Abstract [en]

    Anaphylaxis is a potentially life-threatening condition triggered mainly by the release of inflammatory mediators, notably histamine. In pharmaceutical research, drug discovery, and clinical evaluation, it may be necessary to accurately assess the potential of a compound, event, or disorder to promote the release of histamine. In contrast to the measurement of plasma histamine, determination of the stable metabolite 1-methyl-4-imidazoleacetic acid (tele-MIAA) in urine provides a noninvasive and more reliable methodology to monitor histamine release. This study presents a repeatable high-performance liquid chromatography coupled to electrospray mass spectrometry (LC-ESI-MS) method where tele-MIAA is baseline separated from its structural isomer 1-methyl-5-imidazoleacetic acid (pi-MIAA) and an unknown in human urine. The ion-pairing chromatography method, in reversed-phase mode, based on 0.5 mM tridecafluoroheptanoic acid demonstrated high repeatability and was applied in a clinical development program that comprised a large number of clinical samples from different cohorts. The inter- and intra-run precision of the method for tele-MIAA were 8.4 and 4.3 %, respectively, at the mean urinary concentration level, while method accuracy was between -16.2 and 8.0 % across the linear concentration range of 22-1,111 ng mL(-1). Overall, method precision was greater than that reported in previously published methods and enabled the identification of gender differences that were independent of age or demography. The median concentration measured in female subjects was 3.0 mu mol mmol(-1) of creatinine, and for male subjects, it was 2.1 mu mol mmol(-1) of creatinine. The results demonstrate that the method provides unprecedented accuracy, precision, and practicality for the measurement of tele-MIAA in large clinical settings.

  • 473.
    Kopakkala-Tani, Milla
    et al.
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Elo, Mika
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Sironen, Reijo
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Helminen, Heikki
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    High hydrostatic pressure induces ERK and PI3 kinase phosphorylation in human HCS-2/8 chondrosarcoma cells.2004In: Cellular and Molecular Biology, ISSN 0145-5680, E-ISSN 1165-158X, Vol. 50, no 4, p. 485-490, article id 15529758Article in journal (Refereed)
    Abstract [en]

    High continuous hydrostatic pressure has been shown to affect many cellular functions within the pressurised cells, for instance, accumulation of heat shock protein 70 occurs during pressurisation. Various signal transduction pathways are likely to mediate these changes, however, at the present time our knowledge of the pathways involved is rather limited. The aim of this study was to investigate whether some of the well known transduction pathways are activated by the exposure of human chondrosarcoma cells to 15-30 MPa hydrostatic pressure. The results showed an increased presence of the active, phosphorylated forms of extracellular signal-related kinase (ERK) and phosphoinositide 3-kinase (PI3K) in cells exposed to 15 and 30 MPa continuous hydrostatic pressure, while 0.5 Hz cyclic loading had weaker effects. Inhibition of ERK-pathway with UO126 did not prevent the accumulation of heat shock protein 70. No activation of c-Jun N-terminal protein kinase (JNK) or p38 could be noticed in pressurised cells. In conclusion, we could identify at least two different signal transduction pathways that are activated under high continuous hydrostatic pressure. Accumulation of heat shock protein 70 was independent of ERK-activation.

  • 474. Koshla, Oksana
    et al.
    Yushchuk, Oleksandr
    Stash, Iryna
    Dacyuk, Yuriy
    Myronovskyi, Maksym
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sussmuth, Roderich D.
    Luzhetskyy, Andriy
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Kirsebom, Leif A.
    Ostash, Bohdan
    Gene miaA for post-transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces2019In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 112, no 1, p. 249-265Article in journal (Refereed)
    Abstract [en]

    Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co-regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNA(UAA)(Leu) (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms(2)i(6)A37 residue in most of the A36-A37-containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular.

  • 475.
    Kouokam, J Clavin
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Dobrindt, Ulrich
    Hacker, Jörg
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Active cytotoxic necrotizing factor 1 associated with outer membrane vesicles from uropathogenic Escherichia coli.2006In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 74, no 4, p. 2022-2030Article in journal (Refereed)
    Abstract [en]

    Cytotoxic necrotizing factor type 1 (CNF1) is one of the virulence factors produced by uropathogenic Escherichia coli (UPEC). How this toxin is translocated from the bacterial cytoplasm to the surrounding environment is not well understood. Our data suggest that CNF1 may be regarded as a secreted protein, since it could be detected in culture supernatants. Furthermore, we found that CNF1 was tightly associated to outer membrane vesicles, suggesting that such vesicles play a role in the secretion of this protein. Interestingly, vesicle samples containing CNF1 could exert the effects known for this protein on HeLa cell cultures, showing that CNF1 is transported by vesicles in its active form. Taken together, our results strongly suggest that outer membrane vesicles could be a means for the bacteria to deliver CNF1 to the environment and to the infected tissue. In addition, our results indicate that the histone-like nucleoid structuring protein H-NS has a role in the downregulation of CNF1 production and that it affects the outer membrane vesicle release in UPEC strain J96.

  • 476. Kouremenos, Konstantinos A.
    et al.
    Beale, David J.
    Antti, Henrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Palombo, Enzo A.
    Liquid chromatography time of flight mass spectrometry based environmental metabolomics for the analysis of Pseudomonas putida Bacteria in potable water2014In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 966, p. 179-186Article in journal (Refereed)
    Abstract [en]

    Water supply biofilms have the potential to harbour waterborne diseases, accelerate corrosion, and contribute to the formation of tuberculation in metallic pipes. One particular species of bacteria known to be found in the water supply networks is Pseudomonas sp., with the presence of Pseudomonas putida being isolated to iron pipe tubercles. Current methods for detecting and analysis pipe biofilms are time consuming and expensive. The application of metabolomics techniques could provide an alternative method for assessing biofilm risk more efficiently based on bacterial activity. As such, this paper investigates the application of metabolomic techniques and provides a proof-of-concept application using liquid chromatography coupled with time-of-flight mass spectrometry (LC-ToF-MS) to three biologically independent P. putida samples, across five different growth conditions exposed to solid and soluble iron (Fe). Analysis of the samples in +ESI and -ESI mode yielded 887 and 1789 metabolite features, respectively. Chemometric analysis of the +ESI and -ESI data identified 34 and 39 significant metabolite features, respectively, where features were considered significant if the fold change was greater than 2 and obtained a p-value less than 0.05. Metabolite features were subsequently identified according to the Metabolomics Standard Initiative (MSI) Chemical Analysis Workgroup using analytical standards and standard online LC-MS databases. Possible markers for P. putida growth, with and without being exposed to solid and soluble Fe, were identified from a diverse range of different chemical classes of metabolites including nucleobases, nucleosides, dipeptides, tripeptides, amino acids, fatty acids, sugars, and phospholipids.

  • 477.
    Kovermann, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. University of Konstanz, Department of Chemistry, Constance, Germany.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural basis for ligand binding to an enzyme by a conformational selection pathway2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 24, p. 6298-6303Article in journal (Refereed)
    Abstract [en]

    Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-angstrom X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic side-chains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.

  • 478. Kravchuk, Oksana
    et al.
    Kim, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Klepikov, Pavel
    Parshikov, Alexander
    Georgiev, Pavel
    Savitsky, Mikhail
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia.
    Transvection in Drosophila: trans-interaction between yellow enhancers and promoter is strongly suppressed by a cis-promoter only in certain genomic regions2017In: Chromosoma, ISSN 0009-5915, E-ISSN 1432-0886, Vol. 126, no 3, p. 431-441Article in journal (Refereed)
    Abstract [en]

    Transvection is a phenomenon of interallelic communication whereby enhancers of one allele can activate a promoter located on the homologous chromosome. It has been shown for many independent genes that enhancers preferentially act on the cis-linked promoter, but deletion of this promoter allows the enhancers to act in trans. Here, we tested whether this cis-preference in the enhancer-promoter interaction could be reconstituted outside of the natural position of a gene. The yellow gene was chosen as a model system. Transgenic flies were generated that carried the yellow gene modified by the inclusion of the strategically placed recognition sites for the Cre and Flp recombinases. To facilitate transvection, an endogenous Su(Hw) insulator (1A2) or gypsy insulator was placed behind the yellow gene. Independent action of the recombinases produced a pair of derivative alleles, one containing the promoter-driven yellow gene, and the other, the enhancers and promoter that failed to produce a functional yellow protein. As a result, we observed strong transvection in many genomic regions, suggesting that a complete cis-preference of the enhancer-promoter interactions is mainly restricted to genes in their natural loci.

  • 479.
    Kremnev, Dmitry
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Get in tune: chloroplast and nucleus harmony2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Photosynthetic eukaryots emerged as a result of several billion years of evolution between proeukaryotic cell and ancestral cyanobacteria that formed modern chloroplasts. The symbiotic relationship led to significant rearrangements in the genomes of the plastid and the nucleus: as many as 90 % of all the plastid genes were transferred to the nucleus. The gene transfer has been accompanied by the development of sophisticated regulatory signaling networks originating in the organelle (retrograde) and in the nucleus (anterograde) that coordinate development of the plastid and ensure adequate cell responses to stress signals. In this thesis I have demonstrated that transcriptional activity of PEP in the chloroplast is essential for proper embryo and seedling development in Arabidopsis thaliana. The function of PEP is dependent on the nuclear encoded PEPassociated factor PRIN2 that is able to sense the redox status of the plastid during seedling development and different stress. In response to the plastid status PRIN2 modulates the transcription activity of the PEP enzyme complex. We further established that PRIN2, as an essential component for full PEP activity, is also required to emit the Plastid Gene Expression (PGE) retrograde signal to regulate the Photosynthesis-Associated Nuclear Genes (PhANG) in the nucleus during early seedling growth via GUN1. On the other hand, regulation of PhANG expression during the High Light (HL) conditions requires functional PRIN2 and PEP activity but is GUN1-independent. Another retrograde signal produced by the developing chloroplast is associated with the tetrapyrrole biosynthesis pathway. We have established that accumulation of the chlorophyll intermediate MgProtoIX-ME in the crd mutant triggers repression of the PhANG expression, and this negative signal is mediated by a cytoplasmic protein complex containing the PAPP5 phosphatase. The nuclear targets that receive the tetrapyrrole mediated signal are GLK1 and GLK2 transcription factors that control the PhANG expression and the expression of the enzymes involved in the biosynthesis of chlorophyll.

  • 480.
    Kremnev, Dmitry
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Guinea Diaz, Manuel
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Dubreuil, Carole
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Strand, Åsa
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Redox regulation of PEP activity during seedling development in ArabidopsisManuscript (preprint) (Other academic)
  • 481.
    Kremnev, Dmitry
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Strand, Åsa
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Plastid encoded RNA polymerase activity and expression of photosynthesis genes required for embryo and seed development in Arabidopsis2014In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462XArticle in journal (Refereed)
    Abstract [en]

    Chloroplast biogenesis and function is essential for proper plant embryo and seed development but the molecular mechanisms underlying the role of plastids during embryogenesis are poorly understood. Expression of plastid encoded genes is dependent on two different transcription machineries; a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. However, the division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear. We show here that PLASTID REDOX INSENSITIVE 2 (PRIN2) and CHLOROPLAST STEM-LOOP BINDING PROTEIN 41 kDa (CSP41b), two proteins identified in plastid nucleoid preparations, are essential for proper plant embryo development. Using Co-IP assays and native PAGE we have shown a direct physical interaction between PRIN2 and CSP41b. Moreover, PRIN2 and CSP41b form a distinct protein complex in vitro that binds DNA. The prin2.2 and csp41b-2 single mutants displayed pale phenotypes, abnormal chloroplasts with reduced transcript levels of photosynthesis genes and defects in embryo development. The respective csp41b-2prin2.2 homo/heterozygote double mutants produced abnormal white colored ovules and shrunken seeds. Thus, the csp41b-2prin2.2 double mutant is embryo lethal. In silico analysis of available array data showed that a large number of genes traditionally classified as PEP dependent genes are transcribed during early embryo development from the pre-globular stage to the mature-green-stage. Taken together, our results suggest that PEP activity and consequently the switch from NEP to PEP activity, is essential during embryo development and that the PRIN2-CSP41b DNA binding protein complex possibly is important for full PEP activity during this process.

  • 482. Krypotou, Emilia
    et al.
    Scortti, Mariela
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Oelker, Melanie
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Luisi, Ben F.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Vazquez-Boland, Jose
    Control of Bacterial Virulence through the Peptide Signature of the Habitat2019In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 26, no 7, p. 1815-1827Article in journal (Refereed)
    Abstract [en]

    To optimize fitness, pathogens selectively activate their virulence program upon host entry. Here, we report that the facultative intracellular bacterium Listeria monocytogenes exploits exogenous oligopeptides, a ubiquitous organic N source, to sense the environment and control the activity of its virulence transcriptional activator, PrfA. Using a genetic screen in adsorbent- treated ( PrfA-inducing) medium, we found that PrfA is functionally regulated by the balance between activating and inhibitory nutritional peptides scavenged via the Opp transport system. Activating peptides provide essential cysteine precursor for the PrfA-inducing cofactor glutathione ( GSH). Non-cysteine-containing peptides cause promiscuous PrfA inhibition. Biophysical and co-crystallization studies reveal that peptides inhibit PrfA through steric blockade of the GSH binding site, a regulation mechanism directly linking bacterial virulence and metabolism. L. monocytogenes mutant analysis in macrophages and our functional data support a model in which changes in the balance of antagonistic Oppimported oligopeptides promote PrfA induction intra-cellularly and PrfA repression outside the host.

  • 483.
    Kudahettige Nilsson, Rasika L.
    et al.
    Department of Biotechnology, Luleå Technical University, Luleå, Sweden.
    Holmgren, Marie
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Madavi, Batol
    Nilsson, Robert T.
    Department of Biotechnology, Luleå Technical University, Luleå, Sweden.
    Sellstedt, Anita
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Adaptability of Trametes versicolor to the lignocellulosic inhibitors furfural, HMF, phenol and levulinic acid during ethanol fermentation2016In: Biomass and Bioenergy, ISSN 0961-9534, E-ISSN 1873-2909, Vol. 90, p. 95-100Article in journal (Refereed)
    Abstract [en]

    Ligno-cellulosic biofuels, notably ethanol produced in processes involving biological fermentation, have high potential as renewable alternatives to fossil fuels. However, ligno-cellulose pretreatment procedures generate substances that inhibit current biocatalysts. Thus, efficient methods are required for improving these organisms' tolerance or developing new biocatalysts with higher tolerance to the inhibitors. For this, greater knowledge of the mechanisms involved is needed. Therefore, we examined effects of common inhibitors (phenol, levulinic acid, HMF (hydroxymethylfurfural) and furfural) on growth, utilization of sugars (xylose, mannose and glucose) and enzyme activities of a tolerant organism, the white-rot fungus Trametes versicolor, during 15-day incubations. The fungus metabolized and grew in the presence of all the inhibitors (singly and together) at the applied concentration (0.2–0.6 g/L). When all inhibitors were added, no significant effect of sugar utilization was shown. However, levulinic acid added solely reduced xylose (but not xylose-degrading enzymes) and mannose utilization, but not glucose utilization. Physiological and biotechnological implications of the findings are discussed such as usage of Tversicolor as a detoxifying agent in ethanol production.

  • 484. Kudrin, Pavel
    et al.
    Dzhygyr, Ievgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Ishiguro, Kensuke
    Beljantseva, Jelena
    Maksimova, Elena
    Oliveira, Sofia Raquel Alves
    Varik, Vallo
    Payoe, Roshani
    Konevega, Andrey L.
    Tenson, Tanel
    Suzuki, Tsutomu
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). University of Tartu, Institute of Technology, Tartu, Estonia.
    The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 4, p. 1973-1983Article in journal (Refereed)
    Abstract [en]

    During amino acid starvation the Escherichia coli stringent response factor RelA recognizes deacylated tRNA in the ribosomal A-site. This interaction activates RelA-mediated synthesis of alarmone nucleotides pppGpp and ppGpp, collectively referred to as (p)ppGpp. These two alarmones are synthesized by addition of a pyrophosphate moiety to the 3' position of the abundant cellular nucleotide GTP and less abundant nucleotide GDP, respectively. Using untagged native RelA we show that allosteric activation of RelA by pppGpp increases the efficiency of GDP conversion to achieve the maximum rate of (p) ppGpp production. Using a panel of ribosomal RNA mutants, we show that the A-site finger structural element of 23S rRNA helix 38 is crucial for RelA binding to the ribosome and consequent activation, and deletion of the element severely compromises (p) ppGpp accumulation in E. coli upon amino acid starvation. Through binding assays and enzymology, we show that E. coli RelA does not form a stable complex with, and is not activated by, deacylated tRNA off the ribosome. This indicates that in the cell, RelA first binds the empty A-site and then recruits tRNA rather than first binding tRNA and then binding the ribosome.

  • 485.
    Kumar, Keshav
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Random Initialisation of the Spectral Variables: an Alternate Approach for Initiating Multivariate Curve Resolution Alternating Least Square (MCR-ALS) Analysis2017In: Journal of Fluorescence, ISSN 1053-0509, E-ISSN 1573-4994, Vol. 27, no 6, p. 1957-1968Article in journal (Refereed)
    Abstract [en]

    Multivariate curve resolution alternating least square (MCR-ALS) analysis is the most commonly used curve resolution technique. The MCR-ALS model is fitted using the alternate least square (ALS) algorithm that needs initialisation of either contribution profiles or spectral profiles of each of the factor. The contribution profiles can be initialised using the evolve factor analysis; however, in principle, this approach requires that data must belong to the sequential process. The initialisation of the spectral profiles are usually carried out using the pure variable approach such as SIMPLISMA algorithm, this approach demands that each factor must have the pure variables in the data sets. Despite these limitations, the existing approaches have been quite a successful for initiating the MCR-ALS analysis. However, the present work proposes an alternate approach for the initialisation of the spectral variables by generating the random variables in the limits spanned by the maxima and minima of each spectral variable of the data set. The proposed approach does not require that there must be pure variables for each component of the multicomponent system or the concentration direction must follow the sequential process. The proposed approach is successfully validated using the excitation-emission matrix fluorescence data sets acquired for certain fluorophores with significant spectral overlap. The calculated contribution and spectral profiles of these fluorophores are found to correlate well with the experimental results. In summary, the present work proposes an alternate way to initiate the MCR-ALS analysis.

  • 486.
    Kumar, Rajendra
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Lizana, Ludvig
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Division of CBRN Security and Defence, FOI-Swedish Defence Research Agency, Umeå, Sweden.
    Genomic 3D compartments emerge from unfolding mitotic chromosomes2019In: Chromosoma, ISSN 0009-5915, E-ISSN 1432-0886, Vol. 128, no 1, p. 15-20Article in journal (Refereed)
    Abstract [en]

    The 3D organisation of the genome in interphase cells is not a randomly folded polymer. Rather, experiments show that chromosomes arrange into a network of 3D compartments that correlate with biological processes, such as transcription, chromatin modifications and protein binding. However, these compartments do not exist during cell division when the DNA is condensed, and it is unclear how and when they emerge. In this paper, we focus on the early stages after cell division as the chromosomes start to decondense. We use a simple polymer model to understand the types of 3D structures that emerge from local unfolding of a compact initial state. From simulations, we recover 3D compartments, such as TADs and A/B compartments that are consistently detected in chromosome capture experiments across cell types and organisms. This suggests that the large-scale 3D organisation is a result of an inflation process.

  • 487. Kumar, Ranjeet
    et al.
    Arioz, Candan
    Li, Yaozong
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bosaeus, Niklas
    Rocha, Sandra
    Wittung-Stafshede, Pernilla
    Disease-causing point-mutations in metal-binding domains of Wilson disease protein decrease stability and increase structural dynamics2017In: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 30, no 1, p. 27-35Article in journal (Refereed)
    Abstract [en]

    After cellular uptake, Copper (Cu) ions are transferred from the chaperone Atox1 to the Wilson disease protein (ATP7B) for incorporation into Cu-dependent enzymes in the secretory pathway. Human ATP7B is a large multi-domain membrane-spanning protein which, in contrast to homologues in other organisms, has six similar cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of enzymatic activity and it is thus intriguing that point mutations in MBDs can promote Wilson disease. We here investigated, in vitro and in silico, the biophysical consequences of clinically-observed Wilson disease mutations, G85V in MBD1 and G591D in MBD6, incorporated in domain 4. Because G85 and G591 correspond to a conserved Gly found in all MBDs, we introduced the mutations in the well-characterized MBD4. We found the mutations to dramatically reduce the MBD4 thermal stability, shifting the midpoint temperature of unfolding by more than 20 A degrees C. In contrast to wild type MBD4 and MBD4D, MBD4V adopted a misfolded structure with a large beta-sheet content at high temperatures. Molecular dynamic simulations demonstrated that the mutations increased backbone fluctuations that extended throughout the domain. Our findings imply that reduced stability and enhanced dynamics of MBD1 or MBD6 is the origin of ATP7B dysfunction in Wilson disease patients with the G85V or G591D mutation.

  • 488. Kumar, Vikash
    et al.
    Hainaut, Matthieu
    Delhomme, Nicolas
    Mannapperuma, Chanaka
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Immerzeel, Peter
    Street, Nathaniel
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Henrissat, Bernard
    Mellerowicz, Ewa J.
    Poplar carbohydrate-active enzymes: whole-genome annotation and functional analyses based on RNA expression data2019In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 99, no 4, p. 589-609Article in journal (Refereed)
    Abstract [en]

    Carbohydrate-active enzymes (CAZymes) catalyze the formation and modification of glycoproteins, glycolipids, starch, secondary metabolites and cell wall biopolymers. They are key enzymes for the biosynthesis of food and renewable biomass. Woody biomass is particularly important for long-term carbon storage and as an abundant renewable natural resource for many industrial applications. This study presents a re-annotation of CAZyme genes in the current Populus trichocarpa genome assembly and in silico functional characterization, based on high-resolution RNA-Seq data sets. Altogether, 1914 CAZyme and expansin genes were annotated in 101 families. About 1797 of these genes were found expressed in at least one Populus organ. We identified genes involved in the biosynthesis of different cell wall polymers and their paralogs. Whereas similar families exist in poplar and Arabidopsis thaliana (with the exception of CBM13 found only in poplar), a few families had significantly different copy numbers between the two species. To identify the transcriptional coordination and functional relatedness within the CAZymes and other proteins, we performed co-expression network analysis of CAZymes in wood-forming tissues using the AspWood database () for Populus tremula. This provided an overview of the transcriptional changes in CAZymes during the transition from primary to secondary wall formation, and the clustering of transcripts into potential regulons. Candidate enzymes involved in the biosynthesis of polysaccharides were identified along with many tissue-specific uncharacterized genes and transcription factors. These collections offer a rich source of targets for the modification of secondary cell wall biosynthesis and other developmental processes in woody plants.

  • 489.
    Kunz, Sabine
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Sugar-modulated gene expression and cell division in cell culture and seedlings of A. thaliana2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Throughout their life cycle, plants adjust growth in response to their developmental and environmental situation within the limits of their energetic capacities. This capacity is defined by the local sugar availability, which is constantly modulated through synthesis, transport and consumption of sugar. The monitoring of sugar presence is carried out by a complex signalling network in which simple sugars (e.g. glucose, fructose and sucrose) act as metabolic signals for the modulation of physiological processes. However, often it remains unclear whether the regulation is induced by the simple sugars themselves or by their derivatives generated during sugar metabolism. This thesis focuses on the dissection of distinct sugar signals, their generation, perception and impact on the modulation of gene expression and cell division both in cell culture and young seedlings.

    Based on a stem-cell-like A. thaliana cell culture, which could be sustained in a hormone-free media, a new biological system, supplied with Xyl as the only carbon source was developed. The performance of a variety of sugar and sugar analogue treatments in this novel system allowed for the identification of sugar-responsive candidate genes, which were specifically regulated by glucose, fructose and sucrose. For several genes (e.g. bZIP63, AT5g22920, TPS9, MGD2 and BT2), this regulation required both sugar transport into the cytosol and metabolisation for the generation of the signal. Furthermore, gene expression analyses in young A. thaliana seedlings indicated the requirement for the catalytic activity of hexokinase 1 in the regulation of bZIP63, Atg22920 and BT2 under conditions of a perturbed carbohydrate balance. These findings have been combined in a proposed model for the transcriptional regulation of bZIP63, AT5g22920, TPS9, MGD2 and BT2, which further proposes a function of those genes in the regulation of cell division.

    The optimisation of a protocol for long-term real-time live-cell imaging provided a valuable tool to show that, similar to gene expression, the progression of cell division depended on a sugar-type-specific regulation at the single-cell level; this regulation was most likely caused by prolongation of the interphase. Together with the observation of cell death and growth arrest of the primary root in intact seedlings in response to the glucose analogue 2dog, this led to the conclusion that sugar signals themselves were sufficient to induce cell division. However, the continuation of cell cycle progression and consequently organ growth over long-time required the availability of the energy contained in the sugar.

  • 490.
    Kunz, Sabine
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Gardeström, Per
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Pesquet, Edouard
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Kleczkowski, Leszek
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Hexokinase 1 is required for glucose-induced repression of bZIP63, At5g22920, and BT2 in Arabidopsis2015In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 6, article id 525Article in journal (Refereed)
    Abstract [en]

    Simple sugars, like glucose (Glc) and sucrose (Suc), act as signals to modulate the expression of hundreds of genes in plants. Frequently, however, it remains unclear whether this regulation is induced by the sugars themselves or by their derivatives generated in the course of carbohydrate (CH) metabolism. In the present study, we tested the relevance of different CH metabolism and allocation pathways affecting expression patterns of five selected sugar-responsive genes (bZIP63, At5g22920, BT2, MGD2, and TPS9) in Arabidopsis thaliana. In general, the expression followed diurnal changes in the overall sugar availability. However, under steady growth conditions, this response was hardly impaired in the mutants for CH metabolizing/transporting proteins (adg1, sex1, sus1-4, sus5/6, and tpt2), including also hexokinase1 (HXK1) loss- and gain-of-function plants—gin2.1 and oe3.2, respectively. In addition, transgenic plants carrying pbZIP63::GUS showed no changes in reporter-gene-expression when grown on sugar under steady-state conditions. In contrast, short-term treatments of agar-grown seedlings with 1% Glc or Suc induced pbZIP63::GUS repression, which became even more apparent in seedlings grown in liquid media. Subsequent analyses of liquid-grown gin2.1 and oe3.2 seedlings revealed that Glc -dependent regulation of the five selected genes was not affected in gin2.1, whereas it was enhanced in oe3.2 plants for bZIP63, At5g22920, and BT2. The sugar treatments had no effect on ATP/ADP ratio, suggesting that changes in gene expression were not linked to cellular energy status. Overall, the data suggest that HXK1 does not act as Glc sensor controlling bZIP63, At5g22920, and BT2 expression, but it is nevertheless required for the production of a downstream metabolic signal regulating their expression.

  • 491.
    Kunz, Sabine
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Ménard, Delphine
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Kleczkowski, Leszek
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Pesquet, Edouard
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Monitoring the role of distinct sugars on cell division in Arabidopsis plant cells and seedlingsManuscript (preprint) (Other academic)
    Abstract [en]

    Within the last decades, research on sugar-dependent plant growth provided evidence for a directregulation of cell division by sugars. Recently we showed, in A. thaliana cell suspension cultures, thatdistinct sugars differentially regulate a rapid transcriptional response of genes, some of which functionduring the cell cycle progression. In order to assess the relationship between sugar species and celldivision, we developed a new methodology for long-term real-time live-cell imaging on dividing A.thaliana cell suspension cultures. This technique, using cells grown in hormone-free media, allowed toestimate the cell cycle synchronicity and efficiency of an entire cell population and to monitor thedynamics and geometry of cell division in single cells in response to a given sugars. A marker cell lineconstitutively expressing TUA::GFP, a protein that labels microtubule-based structures hallmarkingprogression of the mitotic division, was used to measure the sugar-dependent efficiency andsynchronicity of the cell cycle progression. Altogether, we were able to confirm the distinct relationshipsof specific sugar molecules on the cell cycle progression at a single cell level. Cell division efficiencyand synchronicity were altered when grown on the different sugars sucrose, glucose and xylose.Interestingly, the progression of the mitotic division appeared unaffected by the sugar species supplied,indicating that length of the interphase is likely to control the cell division rate. In contrast, treatment ofA. thaliana cell cultures and seedlings with the Glc-analogue 2-deoxy-glucose (2dog) led to growtharrest and to cell death during long exposure. The growth resulting from 2dog removal in cell culturesand seedlings showed the unique feature of plants to induce new active zones of cell division.

  • 492. Kusano, Miyako
    et al.
    Fukushima, Atsushi
    Kobayashi, Makoto
    Hayashi, Naomi
    Jonsson, Pär
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Moritz, Thomas
    Ebana, Kaworu
    Saito, Kazuki
    Application of a metabolomic method combining one-dimensional and two-dimensional gas chromatography-time-of-flight/mass spectrometry to metabolic phenotyping of natural variants in rice2007In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 855, no 1, p. 71-79Article in journal (Refereed)
    Abstract [en]

    We have developed a comprehensive method combining analytical techniques of one-dimensional (I D) and two-dimensional (GC x GC) gas chromatography-time-of-flight (TOF)-mass spectrometry. This method was applied to the metabolic phenotyping of natural variants in rice for the 68 world rice core collection (WRC) and two other varieties. Ten metabolites were selected as metabolite representatives, and the selected ion current of each metabolite peak obtained from both techniques were statistically compared. Our method of combining I D- and GC x GC-TOF/MS is useful for the metabolic phenotyping of natural variants in rice for further studies in breeding programs.

  • 493.
    Kuzmenko, Anton
    et al.
    University of Tartu, Institute of Technology ; Molecular Biology Department, Faculty of Biology, M.V. Lomonosov Moscow State University.
    Atkinson, Gemma C
    University of Tartu, Institute of Technology.
    Levitskii, Sergey
    Molecular Biology Department, Faculty of Biology, M.V. Lomonosov Moscow State University.
    Zenkin, Nikolay
    Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom.
    Tenson, Tanel
    University of Tartu, Institute of Technology.
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). University of Tartu, Institute of Technology.
    Kamenski, Piotr
    Molecular Biology Department, Faculty of Biology, M.V. Lomonosov Moscow State University.
    Mitochondrial translation initiation machinery: conservation and diversification2014In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 100C, p. 132-140Article in journal (Refereed)
    Abstract [en]

    The highly streamlined mitochondrial genome encodes almost exclusively a handful of transmembrane components of the respiratory chain complex. In order to ensure the correct assembly of the respiratory chain, the products of these genes must be produced in the correct stoichiometry and inserted into the membrane, posing a unique challenge to the mitochondrial translational system. In this review we describe the proteins orchestrating mitochondrial translation initiation: bacterial-like general initiation factors mIF2 and mIF3, as well as mitochondria-specific components - mRNA-specific translational activators and mRNA-nonspecific accessory initiation factors. We consider how the fast rate of evolution in these organelles has not only created a system that is divergent from that of its bacterial ancestors, but has led to a huge diversity in lineage specific mechanistic features of mitochondrial translation initiation among eukaryotes.

  • 494.
    Kääpä, Eeva
    et al.
    Department of Medical Chemistry, Unversity of Helsinki, Helsinki, Finland.
    Holm, Sten
    Department of Orthopaedics, Sahlgren Hospital, Göteborg, Sweden.
    Inkinen, Ritva
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Tammi, Markku
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Vanharanta, Hannu
    Department of Physical Medicine and Rehabilitation, University of Oulu, Oulu, Finland.
    Proteoglycan chemistry in experimentally injured porcine intervertebral disk.1994In: Journal of spinal disorders, ISSN 0895-0385, E-ISSN 1531-2305, Vol. 7, no 4, p. 296-306, article id 7949697Article in journal (Refereed)
    Abstract [en]

    An animal model of disk degeneration was used to study the concentration levels and types of proteoglycans in the different parts of the intervertebral disk. An annular incision was made with a scalpel blade into the anterior part of the porcine lumbar intervertebral disks via a retroperitoneal approach. Three months after injury the morphology of the injured disk had changed considerably. Disk height was diminished, and in the injured segment osteophytes had formed at the ventral edges of the vertebral body. The nucleus was small, fibrous, and yellowish. The annular lesion had healed by formation of granulation tissue, but the lamellar structure was partially destroyed. The concentration of inorganic [35S]sulfate had decreased across the whole disk, reflecting a decrease in the rate of solute transport. The concentration of incorporated [35S]sulfate had also decreased in the injured disks. The DNA concentration in the anterior annulus and in the nucleus had increased, whereas both the concentration of uronic acid and the ratio of chondroitin-6-sulfate to chondroitin-4-sulfate in the nucleus had decreased. Agarose gel electrophoresis combined with chondroitinase B digestion suggested the presence of dermatan sulfate proteoglycans in the injured annulus fibrosus. The morphology and chemical composition of the disks adjacent to the injured one were normal, and only a slight increase in the concentration of incorporated [35S]sulfate was observed in the disks above the injured one.

  • 495.
    Kääpä, Eeva
    et al.
    Department of Physical, Medicine and Rehabilitation, Helsinki University Hospital, Helsinki, Finland.
    Wang, Wei
    Department of Biology of Physical Activity, University of Jyväskylä, Finland.
    Takala, Timo
    Department of Biology of Physical Activity, University of Jyväskylä, Finland.
    Poussa, Mikko
    Orthopaedic Hospital of the Invalid Foundation, Helsinki, Finland.
    Konttinen, Yrjö
    Fourth Department of Medicine, Helsinki University Hospital, Helsinki, Finland; Department of Biology of Physical Activity, University of Jyväskylä, Finland.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Vanharanta, Heikki
    Department of Physical, Medicine and Rehabilitation, Oulu University, Finland.
    Elevated protein content and prolyl 4-hydroxylase activity in severely degenerated human annulus fibrosus.2000In: Connective Tissue Research, ISSN 0300-8207, E-ISSN 1607-8438, Vol. 41, no 2, p. 93-99, article id 10992155Article in journal (Refereed)
    Abstract [en]

    Alterations involved with the intervertebral disc degeneration are partly well described, however, it is not so well known how collagen network is affected by the disease. We analyzed the rate of collagen biosynthesis (estimated by the enzymic activities of prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase) and the level of hydroxylysylpyridinoline and lysylpyridinoline crosslinks both in normal (n=7) and degenerated (n=7) human annulus fibrosus. The activity of prolyl 4-hydroxylase was significantly increased in degenerated tissue. However, no significant changes in the collagen content or in the amount of hydroxylysylpyridinoline and lysylpyridinoline collagen crosslinks were observed. On the other hand, the content of soluble proteins was significantly increased. Our results suggest that collagen biosynthesis is increased in degenerated human annulus fibrosus, obviously to compensate the impairment of collagen fibers. The faster turnover of collagen in degenerated annulus fibrosus, suggested by the increased prolyl 4-hydroxylase activity and unchanged collagen content, seems not to cause any significant changes in its mature pyridinium crosslink concentrations.

  • 496.
    L. Pourbozorgi, Parham
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Delhomme, Nicolas
    Obi, Ikenna
    Mohammad, Jani Basha
    Sabouri, Nasim
    Topoisomerase 1 and the Pfh1 helicase are both required for proper DNA synthesisManuscript (preprint) (Other academic)
  • 497. L. Pourbozorgi, Parham
    et al.
    Delhomme, Nicolas
    Obi, Ikenna
    Sabouri, Nasim
    Top1 and Pfh1Pif1 maintain the S. pombe mitochondrial genomeManuscript (preprint) (Other academic)
  • 498. Laera, Andreina
    et al.
    Yekta, Sepehr Shakeri
    Hedenström, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Buzier, Remy
    Guibaud, Gilles
    Dario, Mårten
    Esposito, Giovanni
    van Hullebusch, Eric D.
    A simultaneous assessment of organic matter and trace elements bio-accessibility in substrate and digestate from an anaerobic digestion plant2019In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 288, article id 121587Article in journal (Refereed)
    Abstract [en]

    This study evaluates a simultaneous assessment of organic matter (OM) and trace elements (TE) bio-accessibility in substrate and digestate from a full-scale anaerobic digester by a sequential OM extraction method. Simultaneous release of TE was determined along with the extraction of different OM fractions and the effects of extracting reagents on characteristics of OM were evaluated by nuclear magnetic resonance (NMR) spectroscopy. The reagents used for sequential extraction of OM were not enough selective. However, proteins were particularly removed by 0.1 M NaOH, while 72% H2SO4 mainly extracted hemicellulose and cellulose. The OM fractionation allowed for simultaneous extraction of > 60% of total As, Cd, Co, Fe, Mn, Ni and Zn, while the extraction was limited for Al, Cr, Cu, Mo, and Pb. In substrate, > 50% of total As, Co, Mn and Ni and < 40% of total Fe, Zn and Mo were identified in bio-accessible fractions. In digestate, all elements demonstrated poor bio-accessibility except for As.

  • 499. Laitinen, Teresa
    et al.
    Morreel, Kris
    Delhomme, Nicolas
    Gauthier, Adrien
    Schiffthaler, Bastian
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Nickolov, Kaloian
    Brader, Günter
    Lim, Kean-Jin
    Teeri, Teemu H.
    Street, Nathaniel R.
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Boerjan, Wout
    Kärkönen, Anna
    A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism2017In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 174, no 3, p. 1449-1475Article in journal (Refereed)
    Abstract [en]

    Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H2O2) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce (Picea abies) was used as a research model. Scavenging of apoplastic H2O2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including (glycosylated) oligolignols, (glycosylated) flavonoids, and proanthocyanidins, in lignin-forming and H2O2-scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses revealed that scavenging of apoplastic H2O2 resulted in remodulation of the transcriptome, with reduced carbon flux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H2O2 production in addition to potential H2O2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism.

  • 500.
    Lam, Hubert
    et al.
    Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, MA, USA.
    Oh, Dong-Chan
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
    Cava, Felipe
    Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, MA, USA.
    Takacs, Constantin N
    Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, MA, USA.
    Clardy, Jon
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
    de Pedro, Miguel A
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Waldor, Matthew K
    Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, MA, USA.
    D-amino acids govern stationary phase cell wall remodeling in bacteria2009In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 325, no 5947, p. 1552-1555Article in journal (Refereed)
    Abstract [en]

    In all known organisms, amino acids are predominantly thought to be synthesized and used as their L-enantiomers. Here, we found that bacteria produce diverse D-amino acids as well, which accumulate at millimolar concentrations in supernatants of stationary phase cultures. In Vibrio cholerae, a dedicated racemase produced D-Met and D-Leu, whereas Bacillus subtilis generated D-Tyr and D-Phe. These unusual D-amino acids appear to modulate synthesis of peptidoglycan, a strong and elastic polymer that serves as the stress-bearing component of the bacterial cell wall. D-Amino acids influenced peptidoglycan composition, amount, and strength, both by means of their incorporation into the polymer and by regulating enzymes that synthesize and modify it. Thus, synthesis of D-amino acids may be a common strategy for bacteria to adapt to changing environmental conditions.

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