umu.sePublications
Change search
Link to record
Permanent link

Direct link
BETA
Thelander, Lars
Publications (10 of 36) Show all publications
Ranjbarian, F., Vodnala, M., Vodnala, S. M., Rofougaran, R., Thelander, L. & Hofer, A. (2012). Trypanosoma brucei thymidine kinase is tandem protein consisting of two homologous parts, which together enable efficient substrate binding. Journal of Biological Chemistry, 287(21), 17628-17636
Open this publication in new window or tab >>Trypanosoma brucei thymidine kinase is tandem protein consisting of two homologous parts, which together enable efficient substrate binding
Show others...
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 21, p. 17628-17636Article in journal (Refereed) Published
Abstract [en]

Trypanosoma brucei causes African sleeping sickness, a disease for which existing chemotherapies are limited by their toxicity or lack of efficacy. We have found that four parasites, including T. brucei, contain genes where two or four thymidine kinase (TK) sequences are fused into a single open reading frame. The T. brucei full-length enzyme as well as its two constituent parts, domain 1 and domain 2, were separately expressed and characterized. Of potential interest for nucleoside analog development, T. brucei TK was less discriminative against purines than human TK1 with the following order of catalytic efficiencies: thymidine > deoxyuridine ≫ deoxyinosine > deoxyguanosine. Proteins from the TK1 family are generally dimers or tetramers, and the quaternary structure is linked to substrate affinity. T. brucei TK was primarily monomeric but can be considered a two-domain pseudodimer. Independent kinetic analysis of the two domains showed that only domain 2 was active. It had a similar turnover number (k(cat)) as the full-length enzyme but could not self-dimerize efficiently and had a 5-fold reduced thymidine/deoxyuridine affinity. Domain 1, which lacks three conserved active site residues, can therefore be considered a covalently attached structural partner that enhances substrate binding to domain 2. A consequence of the non-catalytic role of domain 1 is that its active site residues are released from evolutionary pressure, which can be advantageous for developing new catalytic functions. In addition, nearly identical 89-bp sequences present in both domains suggest that the exchange of genetic material between them can further promote evolution.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-58608 (URN)10.1074/jbc.M112.340059 (DOI)000306373000061 ()22442154 (PubMedID)
Available from: 2012-09-04 Created: 2012-09-04 Last updated: 2018-06-08Bibliographically approved
Popović-Bijelić, A., Voevodskaya, N., Domkin, V., Thelander, L. & Gräslund, A. (2009). Metal binding and activity of ribonucleotide reductase protein R2 mutants: conditions for formation of the mixed manganese-iron cofactor. Biochemistry, 48(27), 6532-6539
Open this publication in new window or tab >>Metal binding and activity of ribonucleotide reductase protein R2 mutants: conditions for formation of the mixed manganese-iron cofactor
Show others...
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 27, p. 6532-6539Article in journal (Refereed) Published
Abstract [en]

Class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (C. tm.) lacks the tyrosyl radical and uses a Mn(IV)-Fe(III) cluster for cysteinyl radical initiation in the large subunit. Here we investigated and compared the metal content and specific activity of the C. tm. wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli. Our results indicate that the affinity of the RNR R2 proteins for binding metals is determined by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins. In mouse R2, this tyrosyl residue is crucial for the activity of the enzyme, but in C. tm., the corresponding phenylalanine plays no obvious role in activation or catalysis. However, for the C. tm. wild-type R2 protein to bind Mn and gain high specific activity, there seems to be a strong preference for F over Y at this position. In studies of mouse RNR, we find that the native R2 protein does not bind Mn whereas its Y177F mutant incorporates a significant amount of Mn and exhibits 1.4% of native mouse RNR activity. The observation suggests that a manganese-iron cofactor is associated with the weak activity in this protein.

Identifiers
urn:nbn:se:umu:diva-42882 (URN)10.1021/bi900693s (DOI)19492792 (PubMedID)
Available from: 2011-04-14 Created: 2011-04-14 Last updated: 2018-06-08Bibliographically approved
Pontarin, G., Fijolek, A., Pizzo, P., Ferraro, P., Rampazzo, C., Pozzan, T., . . . Bianchi, V. (2008). Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage.. Proceedings of the National Academy of Sciences of the United States of America, 105(46), 17801-17806
Open this publication in new window or tab >>Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage.
Show others...
2008 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 46, p. 17801-17806Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and repair. The mammalian enzyme consists of a catalytic (R1) and a radical-generating (R2 or p53R2) subunit. During S-phase, a R1/R2 complex is the major provider of deoxynucleotides. p53R2 is induced by p53 after DNA damage and was proposed to supply deoxynucleotides for DNA repair after translocating from the cytosol to the cell nucleus. Similarly R1 and R2 were claimed to move to the nucleus during S-phase to provide deoxynucleotides for DNA replication. These models suggest translocation of ribonucleotide reductase subunits as a regulatory mechanism. In quiescent cells that are devoid of R2, R1/p53R2 synthesizes deoxynucleotides also in the absence of DNA damage. Mutations in human p53R2 cause severe mitochondrial DNA depletion demonstrating a vital function for p53R2 different from DNA repair and cast doubt on a nuclear localization of the protein. Here we use three independent methods to localize R1, R2, and p53R2 in fibroblasts during cell proliferation and after DNA damage: Western blotting after separation of cytosol and nuclei; immunofluorescence in intact cells; and transfection with proteins carrying fluorescent tags. We thoroughly validate each method, especially the specificity of antibodies. We find in all cases that ribonucleotide reductase resides in the cytosol suggesting that the deoxynucleotides produced by the enzyme diffuse into the nucleus or are transported into mitochondria and supporting a primary function of p53R2 for mitochondrial DNA replication.

Place, publisher, year, edition, pages
National Academy of Sciences, 2008
Keywords
DNA precursors, immunofluorescence, mitochondrial DNA, p53R2, subcellular localization
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-22411 (URN)10.1073/pnas.0808198105 (DOI)18997010 (PubMedID)
Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2018-06-08Bibliographically approved
Fijolek, A., Hofer, A. & Thelander, L. (2007). Expression, purification, characterization, and in vivo targeting of trypanosome CTP synthetase for treatment of African sleeping sickness.. Journal of biological chemistry, 282(16), 11858-11865
Open this publication in new window or tab >>Expression, purification, characterization, and in vivo targeting of trypanosome CTP synthetase for treatment of African sleeping sickness.
2007 (English)In: Journal of biological chemistry, ISSN 0021-9258, Vol. 282, no 16, p. 11858-11865Article in journal (Refereed) Published
Keywords
Animals, Carbon-Nitrogen Ligases/*biosynthesis/*chemistry, Cytidine/chemistry, Cytosine/chemistry, Dose-Response Relationship; Drug, Enzyme Inhibitors/pharmacology, Humans, Kinetics, Male, Mice, Mice; Inbred C57BL, Trypanocidal Agents/pharmacology, Trypanosoma brucei brucei/*enzymology, Trypanosoma brucei gambiense/*enzymology, Trypanosomiasis; African/*therapy
Identifiers
urn:nbn:se:umu:diva-6341 (URN)17331943 (PubMedID)
Available from: 2007-12-09 Created: 2007-12-09 Last updated: 2018-06-09Bibliographically approved
Pontarin, G., Ferraro, P., Håkansson, P., Thelander, L., Reichard, P. & Bianchi, V. (2007). p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage. Journal of Biological Chemistry, 282(23), 16820-8
Open this publication in new window or tab >>p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage
Show others...
2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 282, no 23, p. 16820-8Article in journal (Refereed) Published
Keywords
Blotting; Western, Cell Cycle Proteins/*physiology, Cell Line, DCMP Deaminase/metabolism, DNA Damage, DNA Repair, Deoxyribonucleotides/*metabolism, Humans, Ribonucleotide Reductases/*physiology, Thymidylate Synthase/metabolism
Identifiers
urn:nbn:se:umu:diva-5953 (URN)doi:10.1074/jbc.M701310200 (DOI)17416930 (PubMedID)
Available from: 2007-12-04 Created: 2007-12-04 Last updated: 2018-06-09Bibliographically approved
Thelander, L. (2007). Ribonucleotide reductase and mitochondrial DNA synthesis.. Nature. Genetics, 39(6), 703-4
Open this publication in new window or tab >>Ribonucleotide reductase and mitochondrial DNA synthesis.
2007 (English)In: Nature. Genetics, ISSN 1061-4036, Vol. 39, no 6, p. 703-4Article in journal (Refereed) Published
Keywords
Animals, Cell Cycle Proteins/*genetics, DNA; Mitochondrial/*biosynthesis/*genetics, Gene Deletion, Humans, Mitochondria; Muscle/*genetics, Mutation/*genetics, Ribonucleotide Reductases/*genetics, Tumor Suppressor Protein p53/genetics/*metabolism
Identifiers
urn:nbn:se:umu:diva-6340 (URN)17534360 (PubMedID)
Available from: 2007-12-09 Created: 2007-12-09 Last updated: 2018-06-09Bibliographically approved
Voevodskaya, N., Narvaez, A.-J., Domkin, V., Torrents, E., Thelander, L. & Gräslund, A. (2006). Chlamydial ribonucleotide reductase: tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster.. Proceedings of the National Academy of Sciences of the United States of America, 103(26), 9850-4
Open this publication in new window or tab >>Chlamydial ribonucleotide reductase: tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster.
Show others...
2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, Vol. 103, no 26, p. 9850-4Article in journal (Refereed) Published
Keywords
Bacterial Proteins/*chemistry, Catalysis, Chlamydia trachomatis/*enzymology, Electron Spin Resonance Spectroscopy, Free Radicals/chemistry, Iron/*chemistry, Oxidation-Reduction, Ribonucleotide Reductases/*chemistry, Tyrosine/*chemistry
Identifiers
urn:nbn:se:umu:diva-6869 (URN)16777966 (PubMedID)
Available from: 2007-12-20 Created: 2007-12-20 Last updated: 2018-06-09Bibliographically approved
Håkansson, P., Hofer, A. & Thelander, L. (2006). Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells.. Journal of Biological Chemistry, 281(12), 7834-7841
Open this publication in new window or tab >>Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells.
2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 12, p. 7834-7841Article in journal (Refereed) Published
Keywords
Animals, Catalysis, Cell Cycle, DNA/chemistry/metabolism, DNA Damage, DNA Repair, DNA Replication, DNA; Mitochondrial/metabolism, Deoxyribonucleotides/*chemistry, Dose-Response Relationship; Drug, Fibroblasts/metabolism, Flow Cytometry, Free Radicals, G0 Phase, G1 Phase, Hydroxyurea/chemistry, Immunoblotting, Mice, Mice; Inbred BALB C, Mitochondria/metabolism, NIH 3T3 Cells, Protein Binding, Recombinant Proteins/chemistry, Ribonucleotide Reductases/chemistry, Ribonucleotides/*chemistry, S Phase, Saccharomycetales, Time Factors, Up-Regulation
Identifiers
urn:nbn:se:umu:diva-6343 (URN)10.1074/jbc.M512894200 (DOI)16436374 (PubMedID)
Available from: 2007-12-09 Created: 2007-12-09 Last updated: 2018-06-09Bibliographically approved
Narváez, A. J., Voevodskaya, N., Thelander, L. & Gräslund, A. (2006). The involvement of Arg265 of mouse ribonucleotide reductase R2 protein in proton transfer and catalysis.. Journal of biological chemistry, 281(36), 26022-8
Open this publication in new window or tab >>The involvement of Arg265 of mouse ribonucleotide reductase R2 protein in proton transfer and catalysis.
2006 (English)In: Journal of biological chemistry, ISSN 0021-9258, Vol. 281, no 36, p. 26022-8Article in journal (Refereed) Published
Keywords
Animals, Arginine/*chemistry, Electron Transport/*physiology, Iron/metabolism, Mice, Models; Molecular, Molecular Sequence Data, Mutagenesis; Site-Directed, Oxygen/metabolism, Protein Structure; Tertiary, Protein Subunits/chemistry/genetics/metabolism, Protons, Ribonucleotide Reductases/chemistry/genetics/metabolism, Surface Plasmon Resonance
Identifiers
urn:nbn:se:umu:diva-6342 (URN)16829694 (PubMedID)
Available from: 2007-12-09 Created: 2007-12-09 Last updated: 2018-06-09Bibliographically approved
Håkansson, P., Dahl, L., Chilkova, O., Domkin, V. & Thelander, L. (2006). The Schizosaccharomyces pombe replication inhibitor Spd1 regulates ribonucleotide reductase activity and dNTPs by binding to the large Cdc22 subunit.. Journal of Biological Chemistry, 281(3), 1778-1783
Open this publication in new window or tab >>The Schizosaccharomyces pombe replication inhibitor Spd1 regulates ribonucleotide reductase activity and dNTPs by binding to the large Cdc22 subunit.
Show others...
2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 3, p. 1778-1783Article in journal (Refereed) Published
Keywords
Cell Cycle Proteins/genetics/*metabolism/*physiology, Cloning; Molecular, DNA Replication, Deoxyribonucleotides/*metabolism, Escherichia coli/genetics, G1 Phase, Kinetics, Protein Subunits/metabolism, Recombinant Proteins/isolation & purification/metabolism, Ribonucleotide Reductases/antagonists & inhibitors/genetics/*metabolism, Schizosaccharomyces/cytology/enzymology/*genetics, Schizosaccharomyces pombe Proteins/genetics/*metabolism/*physiology
Identifiers
urn:nbn:se:umu:diva-6344 (URN)10.1074/jbc.M511716200 (DOI)16317005 (PubMedID)
Available from: 2007-12-09 Created: 2007-12-09 Last updated: 2018-06-09Bibliographically approved
Organisations

Search in DiVA

Show all publications