umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Anders Hofer)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs.

T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease.

It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice.

The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open reading frame. The recombinant T. brucei TK, which belongs to the TK1 family, showed broad substrate specificity. The enzyme phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine, as well as the purine nucleosides deoxyinosine and deoxyguanosine. When the repeated sequences of the tandem TbTK were expressed individually as domains, only domain 2 was active. However, the protein could not dimerize and had a 5-fold reduced affinity to its pyrimidine substrates but a similar turnover number as the full-length enzyme. The expressed domain 1 was inactive and sequence analysis revealed that some active residues, which are needed for substrate binding and catalysis, are absent. Generally, the TK1 family enzymes form dimers or tetramers and the quaternary structure is linked to the affinity for the substrates. The covalently linked inactive domain-1 helps domain-2 to form a pseudodimer for the efficient binding of substrates. In addition, we discovered a repetition of an 89-bp sequence in both domain 1 and domain 2, which suggests a genetic exchange between the two domains.

T. brucei is very dependent on de novo synthesis via ribonucleotide reductase (RNR) for the production of dNTPs. Even though T. brucei RNR belongs to the class Ia RNR family and contains an ATP-binding cone, it lacks inhibition by dATP. The mechanism behind the RNR activation by ATP and inactivation by dATP was a puzzle for a long time in the ~50 years of RNR research. We carried out oligomerization studies on mouse and E. coli RNRs, which belongs to the same family as T. brucei, to get an understanding of the molecular mechanism behind overall activity regulation. We found that the oligomerization status of RNRs and overall activity mechanism are interlinked with each other. 

Place, publisher, year, edition, pages
Umeå: Umeå University , 2013. , 48 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1524
Keyword [en]
Trypanosoma brucei, adenosine kinase, thymidine kinase, methylthioadenosine phosphorylase, mouser ribonucleotide reductase, E. coli ribonucleotide reductase, RNR, Ara-A, F-Ara-A, dNTP, NTP, doexynucleotide metabolism, nucleosides, nucleoside kinases, allosteric regulation
National Category
Biochemistry and Molecular Biology
Research subject
biological chemistry
Identifiers
URN: urn:nbn:se:umu:diva-80904ISBN: 978-91-7459-737-0 (print)OAI: oai:DiVA.org:umu-80904DiVA: diva2:651918
Public defence
2013-11-08, KB3A9, KBC-huset,, Umeå, 10:00 (English)
Opponent
Supervisors
Projects
Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.
Funder
Swedish Research CouncilSida - Swedish International Development Cooperation Agency
Available from: 2013-10-18 Created: 2013-09-27 Last updated: 2013-10-18Bibliographically approved
List of papers
1. Adenosine kinase mediates high affinity adenosine salvage in Trypanosoma brucei
Open this publication in new window or tab >>Adenosine kinase mediates high affinity adenosine salvage in Trypanosoma brucei
Show others...
2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 9, 5380-5388 p.Article in journal (Refereed) Published
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-9175 (URN)10.1074/jbc.M705603200 (DOI)18167353 (PubMedID)
Available from: 2008-03-07 Created: 2008-03-07 Last updated: 2013-10-18Bibliographically approved
2. The protective role of Trypanosoma brucei methylthioadenosine phosphorylase against deoxyadenosine and deoxyadenosine analogs
Open this publication in new window or tab >>The protective role of Trypanosoma brucei methylthioadenosine phosphorylase against deoxyadenosine and deoxyadenosine analogs
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-81610 (URN)
Available from: 2013-10-18 Created: 2013-10-18 Last updated: 2013-10-18Bibliographically approved
3. Trypanosoma brucei thymidine kinase is tandem protein consisting of two homologous parts, which together enable efficient substrate binding
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, 17628-17636 p.Article 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: 2017-12-07Bibliographically approved
4. Enzymatically active mammalian ribonucleotide reductase exists primarily as an α6β2 octamer
Open this publication in new window or tab >>Enzymatically active mammalian ribonucleotide reductase exists primarily as an α6β2 octamer
2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 38, 27705-27711 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase synthesizes deoxyribonucleotides, which are essential building blocks for DNA synthesis. The mammalian ribonucleotide reductase is described as an alpha(2)beta(2) complex consisting of R1 (alpha) and R2 (beta) proteins. ATP stimulates and dATP inhibits enzyme activity by binding to an allosteric site called the activity site on the R1 protein. Despite the opposite effects by ATP and dATP on enzyme activity, both nucleotides induce formation of R1 oligomers. By using a new technique termed Gas-phase Electrophoretic-Mobility Macromolecule Analysis (GEMMA), we have found that the ATP/dATP-induced R1 oligomers have a defined size (hexamers) and can interact with the R2 dimer to form an enzymatically active protein complex (alpha(6)beta(2)). The newly discovered alpha(6)beta(2) complex can either be in an active or an inhibited state depending on whether ATP or dATP is bound. Our results suggest that this protein complex is the major form of ribonucleotide reductase at physiological levels of R1-R2 protein and nucleotides.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-22397 (URN)10.1074/jbc.M605573200 (DOI)16861739 (PubMedID)
Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2013-10-18Bibliographically approved
5. Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase
Open this publication in new window or tab >>Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase
Show others...
2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 51, 35310-35318 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) is a key enzyme for the synthesis of the four DNA building blocks. Class Ia RNRs contain two subunits, denoted R1 (α) and R2 (β). These enzymes are regulated via two nucleotide-binding allosteric sites on the R1 subunit, termed the specificity and overall activity sites. The specificity site binds ATP, dATP, dTTP, or dGTP and determines the substrate to be reduced, whereas the overall activity site binds dATP (inhibitor) or ATP. By using gas-phase electrophoretic mobility macromolecule analysis and enzyme assays, we found that the Escherichia coli class Ia RNR formed an inhibited α4β4 complex in the presence of dATP and an active α2β2 complex in the presence of ATP (main substrate: CDP), dTTP (substrate: GDP) or dGTP (substrate: ADP). The R1-R2 interaction was 30–50 times stronger in the α4β4 complex than in the α2β2complex, which was in equilibrium with free α2 and β2 subunits. Studies of a known E. coli R1 mutant (H59A) showed that deficient dATP inhibition correlated with reduced ability to form α4β4 complexes. ATP could also induce the formation of a generally inhibited α4β4 complex in the E. coli RNR but only when used in combination with high concentrations of the specificity site effectors, dTTP/dGTP. Both allosteric sites are therefore important for α4β4 formation and overall activity regulation. The E. coli RNR differs from the mammalian enzyme, which is stimulated by ATP also in combination with dGTP/dTTP and forms active and inactive α6β2 complexes.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2008
Keyword
E. coli ribonucleotide reductase, RNR, oligomerization, octamer, ATP, dATP
National Category
Biochemistry and Molecular Biology
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-10704 (URN)10.1074/jbc.M806738200 (DOI)18835811 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2008-10-23 Created: 2008-10-23 Last updated: 2014-01-22Bibliographically approved

Open Access in DiVA

fulltext(27792 kB)565 downloads
File information
File name FULLTEXT02.pdfFile size 27792 kBChecksum SHA-512
a2cbed4e1831b0d98d1311038fad022de149260d0181ab959a9a71f27d6198e1dbe93b7a71c45022119f9e2d3f4528cc5bfb73039415d2d28d625437989bfa86
Type fulltextMimetype application/pdf

Authority records BETA

Vodnala, Munender

Search in DiVA

By author/editor
Vodnala, Munender
By organisation
Department of Medical Biochemistry and Biophysics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 566 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 938 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf